LNA antagonists targeting the androgen receptor

ABSTRACT

The invention relates to oligonucleotide compounds (oligomers), which target androgen receptor mRNA in a cell, leading to reduced expression of the androgen receptor. Reduction of androgen receptor expression is beneficial for the treatment of certain disorders, such as hyperproliferative disorders (e.g., cancer). The invention provides therapeutic compositions comprising oligomers and methods for modulating the expression of androgen receptor using said oligomers, including methods of treatment.

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application Ser. No. 60/990,125 filed Nov. 26, 2007, thedisclosure of which is incorporated herein by reference in its entirety.

FIELD OF INVENTION

The invention provides compounds, compositions and methods formodulating the expression of the androgen receptor. In particular, thisinvention relates to oligomeric compounds (oligomers), which target theandrogen receptor mRNA in a cell, leading to reduced expression of theandrogen receptor. Reduction of androgen receptor expression isbeneficial for a range of medical disorders, such as cancer,particularly prostate cancer or breast cancer.

BACKGROUND

The androgen receptor (“AR”) is a type of nuclear receptor which isactivated by binding of either of the androgenic hormones testosteroneor dihydrotestosterone. The main function of the androgen receptor is asa DNA binding transcription factor which regulates gene expression.However the androgen receptor also has additional functions independentof DNA binding. The androgen receptor is most closely related to theprogesterone receptor, and progestins in higher dosages can block theandrogen receptor.

Whilst in humans the AR gene is single copy and found on the Xchromosome at position Xq11-12, the receptor itself exists in twoiso-forms (A and B). AR-A is an 87 kDa protein which lacks the first 187amino acids (N-terminal truncation). Isoform AR-B is the full length 110kDa version.

The binding of an androgen to the androgen receptor induces aconformational change in the receptor, resulting in a dissociation ofheat shock proteins, dimerization and transport from the cytosol to thecell nucleus where the androgen receptor dimer binds to specific DNAsequences—referred to as hormone response elements. Depending on theinteraction with other nuclear proteins, the AR controls geneexpression, either increasing or decreasing transcription of specificgenes, such as insulin-like growth factor I (IGF-1).

Androgen receptors can also have cytoplasmic activities throughinteraction with signal transduction proteins in the cytoplasm. Androgenbinding to cytoplasmic androgen receptors can cause rapid changes incell function independent of gene transcription, for example iontransport, as well as indirect influence of gene transcription, forexample via mediating other signal transduction pathways, therebyinfluencing the activity of other transcription factors.

The over-expression of androgen receptor, or expression of mutatedandrogen receptor genes, has been indicated in several diseases, such ascancer, including prostate cancer and breast cancer, as well as otherdisorders such as polyglutamate disease (Monks et al., PNAS Nov. 2,2007, published on line) alopecia, benign prostatic hyperplasia, spinaland muscular atrophy and Kennedy disease.

WO97/11170 describes a method of treating a patient diagnosed as havingbenign prostatic hyperplasia or a prostate cancer comprisingadministering an antisense oligonucleotide which selectively hybridisesto the androgen receptor mRNA. Three antisense oligonucleotide sequencesof between 27-29 nucleotides are disclosed.

U.S. Pat. No. 6,733,776 and EP 0 692 972 describe a method for treatingandrogenic alopecia by applying liposomes comprising an antisensenucleic acid that hybridises to an androgen receptor gene. No antisensemolecules having specific sequences and targeting the androgen receptorare provided.

US 2005/0164970 describes a method of treating prostate cancer usingsiRNA complexes targeting the androgen receptor mRNA.

WO 2005/027833 describes a method of treating prostate cancer comprisingadministering to a patient an oligonucleotide comprising between 12-40morpholino sub-units.

WO 2001/083740 describes an antisense compound having an unchargedmorpholino backbone of between 18 to 20 contiguous units which targetsthe human androgen receptor.

Morpholino antisense compounds work via binding to the nucleic acidtarget to block access to the mRNA by other molecules, such as moleculesinvolved in mRNA splicing or translation initiation.

U.S. Pat. No. 7,067,256 describes a ribozyme which apparently mediatesinactivation of the androgen receptor. A 19-nucleotide RNA antisensemolecule targeted to a region of the androgen receptor mRNA is provided.

However, despite the application of siRNA, morpholino-containingantisense oligonucleotides and ribozymes, none of the above androgenreceptor inhibitors have been successful in efficiently down-regulatingthe androgen-receptor in vivo and at pharmacologically acceptabledosages.

The invention provides a new class of androgen receptor antagonistswhich contain locked nucleic acid (“LNA”) monomers, and are targeted toparticularly effective target sites on the androgen receptor mRNA.

SUMMARY OF INVENTION

The invention provides an oligomer of from 10-50 monomers, such as 10-30monomers which comprises a first region of 10-50 monomers, such as 10-30monomers, wherein the sequence of the first region is at least 80%(e.g., 85%, 90%, 95%, 98%, or 99%) identical to the reverse complementof a target region of a nucleic acid which encodes a mammalian androgenreceptor, such as a mammalian androgen receptor gene or mRNA, such as anucleic acid having the sequence set forth in SEQ ID NO: 1, or naturallyoccurring variants thereof. Thus, for example, the oligomer hybridizesto a region of a single-stranded nucleic acid molecule having thesequence shown in SEQ ID NO: 1.

The invention provides for a conjugate comprising the oligomer accordingto the invention, and at least one non-nucleotide or non-polynucleotidemoiety covalently attached to the oligomer.

The invention provides for a pharmaceutical composition comprising theoligomer or the conjugate according to the invention, and apharmaceutically acceptable diluent, carrier, salt or adjuvant.

The invention provides for the oligomer or the conjugate according tothe invention, for use as a medicament, such as for the treatment of adisease or a medical disorder as disclosed herein, such as ahyperproliferative disorder, such as cancer or other hyperproliferativedisorder. The invention provides for the use of an oligomer or theconjugate according to the invention, for the manufacture of amedicament for the treatment of a disease or disorder as disclosedherein, such as a hyperproliferative disorder, such as cancer.

The invention provides for a method of treating a disease or disorder asdisclosed herein, such as a hyperproliferative disorder, such as cancer,the method comprising administering an oligomer, a conjugate or apharmaceutical composition according to the invention to a patientsuffering from or susceptible to the disease or disorder.

The invention provides for a method for the inhibition of androgenreceptor in a cell which is expressing androgen receptor, the methodcomprising administering an oligomer, or a conjugate according to theinvention to the cell so as to effect the inhibition of androgenreceptor expression in said cell.

The invention provides an oligomer of from 10-50 monomers, whichcomprises a first region of 10-50 contiguous monomers, wherein the basesequence is at least 80% identical to the reverse complement of a targetregion of a nucleic acid which encodes a mammalian androgen receptor.

The invention further provides a conjugate comprising the oligomeraccording to the invention, which comprises at least one non-nucleotideor non-polynucleotide moiety (“conjugated moiety”) covalently attachedto the oligomer of the invention.

The invention provides for pharmaceutical compositions comprising anoligomer or conjugate of the invention, and a pharmaceuticallyacceptable diluent, carrier, salt or adjuvant.

The invention further provides for an oligomer according to theinvention, for use in medicine.

The invention further provides for the use of the oligomer of theinvention for the manufacture of a medicament for the treatment of oneor more of the diseases referred to herein, such as a disease selectedfrom the group consisting of cancer, such as breast cancer or prostatecancer, alopecia, benign prostatic hyperplasia, spinal and muscularatrophy, Kennedy disease and polyglutamate disease.

The invention further provides for an oligomer according to theinvention, for use for the treatment of one or more of the diseasesreferred to herein, such as a disease selected from the group consistingof cancer, such as breast cancer or prostate cancer, alopecia, benignprostatic hyperplasia, spinal and muscular atrophy, Kennedy disease andpolyglutamate disease.

Pharmaceutical and other compositions comprising an oligomer of theinvention are also provided. Further provided are methods ofdown-regulating the expression of AR in cells or tissues comprisingcontacting said cells or tissues, in vitro or in vivo, with one or moreof the oligomers, conjugates or compositions of the invention.

Also disclosed are methods of treating a non-human animal or a humansuspected of having, or susceptible to, a disease or condition,associated with expression, or over-expression of AR by administering tothe animal or human a therapeutically or prophylactically effectiveamount of one or more of the oligomers, conjugates or pharmaceuticalcompositions of the invention. Further, methods of using oligomers forthe inhibition of expression of AR, and for treatment of diseasesassociated with activity of AR are provided.

The invention provides for a method for treating a disease selected fromthe group consisting of: cancer, such as breast cancer or prostatecancer, alopecia, benign prostatic hyperplasia, spinal and muscularatrophy, Kennedy disease and polyglutamate disease, the methodcomprising administering an effective amount of one or more oligomers,conjugates, or pharmaceutical compositions thereof to a patient in needthereof.

The invention provides for methods of inhibiting (e.g., bydown-regulating) the expression of AR in a cell or a tissue, the methodcomprising the step of contacting the cell or tissue with an effectiveamount of one or more oligomers, conjugates, or pharmaceuticalcompositions thereof, to effect down-regulation of expression of AR.

BRIEF DESCRIPTION OF FIGURES

FIG. 1. Oligonucleotides presented in Table 3 were evaluated for theirpotential to knockdown the androgen receptor mRNA at concentrations of1, 4 and 16 n-M in MCF7 cells 24 hours after transfection usingReal-time PCR. All results were normalised to GAPDH and inhibition of ARmRNA is shown as percent of untreated control. Results shown are anaverage of three independent experiments.

FIG. 2. Oligonucleotides presented in Table 3 were evaluated for theirpotential to knockdown the androgen receptor mRNA at concentrations of1, 4 and 16 nM in A549 cells 24 hours after transfection using Real-timePCR. All results were normalised to GAPDH and inhibition of AR mRNA isshown as percent of untreated control. Results shown are an average ofthree independent experiments.

FIG. 3. Sequence alignment of the human Androgen receptor mRNA sequence(GenBank Accession No.: NM_(—)000044; SEQ ID NO: 1) and the mouseAndrogen receptor mRNA sequence (GenBank Accession No.: NM_(—)013476;SEQ ID NO: 81).

FIG. 4. Location of presently preferred target regions of the human ARmRNA (cDNA; SEQ ID NO: 1) targeted by oligomers according to theinvention. Although 16mer target sites have been shown, in someembodiments these target regions comprise an additional 4 monomers 5′ or3′ to the target regions shown—i.e. are target regions comprising up to24 contiguous monomers.

FIG. 5. SEQ ID NO: 1 Homo sapiens androgen receptor (dihydrotestosteronereceptor; testicular feminization; spinal and bulbar muscular atrophy;Kennedy disease) (AR), transcript variant 1, mRNA. (GenBank Accessionnumber: NM_(—)000044).

FIG. 6. SEQ ID NO 81: Mouse androgen receptor mRNA sequence.

FIG. 7. SEQ ID NO 82: Rhesus monkey androgen receptor mRNA sequence.

FIG. 8. SEQ ID NO 83: Homo sapiens androgen receptor protein amino acidsequence.

FIG. 9. SEQ ID NO 84: Mouse androgen receptor protein amino acidsequence.

FIG. 10. SEQ ID NO 85: Rhesus monkey androgen receptor protein aminoacid sequence.

FIG. 11: AR mRNA in LNCaP, 24 h post-transfection

FIG. 12: AR mRNA in A549, 24 h post-transfection

FIG. 13: Cell proliferation assay—A549, time course post-transfection

FIG. 14: Cell proliferation assay—time course post-transfection

FIG. 15: Caspase 3/7 activity in LNCaP cells, 24, 48 or 72 hourspost-transfection.

FIG. 16: Caspase 3/7 activity in A549 cells, 24, 48 or 72 hourspost-transfection

FIG. 17: Average PSA in plasma after in vivo oligomer treatment.

FIG. 18: In vivo inhibition of tumor growth

DETAILED DESCRIPTION OF INVENTION The Oligomer

The invention employs oligomeric compounds (referred herein asoligomers), for use in modulating the function of nucleic acid moleculesencoding mammalian androgen receptor, such as the androgen receptornucleic acid shown in SEQ ID NO: 1, and naturally occurring variants ofsuch nucleic acid molecules encoding mammalian androgen receptor. Theterm “oligomer” in the context of the invention, refers to a moleculeformed by covalent linkage of two or more monomers (i.e. anoligonucleotide). In some embodiments, the oligomer comprises orconsists of from 10-30 covalently linked monomers.

The term “monomer” includes both nucleosides and deoxynucleosides(collectively, “nucleosides”) that occur naturally in nucleic acids andthat do not contain either modified sugars or modified nucleobases,i.e., compounds in which a ribose sugar or deoxyribose sugar iscovalently bonded to a naturally-occurring, unmodified nucleobase (base)moiety (i.e., the purine and pyrimidine heterocycles adenine, guanine,cytosine, thymine or uracil) and “nucleoside analogues,” which arenucleosides that either do occur naturally in nucleic acids or do notoccur naturally in nucleic acids, wherein either the sugar moiety isother than a ribose or a deoxyribose sugar (such as bicyclic sugars or2′ modified sugars, such as 2′ substituted sugars), or the base moietyis modified (e.g., 5-methylcytosine), or both.

An “RNA monomer” is a nucleoside containing a ribose sugar and anunmodified nucleobase.

A “DNA monomer” is a nucleoside containing a deoxyribose sugar and anunmodified nucleobase.

A “Locked Nucleic Acid monomer,” “locked monomer,” or “LNA monomer” is anucleoside analogue having a bicyclic sugar, as further described hereinbelow.

The terms “corresponding nucleoside analogue” and “correspondingnucleoside” indicate that the base moiety in the nucleoside analogue andthe base moiety in the nucleoside are identical. For example, when the“nucleoside” contains a 2-deoxyribose sugar linked to an adenine, the“corresponding nucleoside analogue” contains, for example, a modifiedsugar linked to an adenine base moiety.

The terms “oligomer,” “oligomeric compound,” and “oligonucleotide” areused interchangeably in the context of the invention, and refer to amolecule formed by covalent linkage of two or more contiguous monomersby, for example, a phosphate group (forming a phosphodiester linkagebetween nucleosides) or a phosphorothioate group (forming aphosphorotioate linkage between nucleosides). The oligomer consists of,or comprises, 10-50 monomers, such as 10-30 monomers.

In some embodiments, an oligomer comprises nucleosides, or nucleosideanalogues, or mixtures thereof as referred to herein. An “LNA oligomer”or “LNA oligonucleotide” refers to an oligonucleotide containing one ormore LNA monomers.

Nucleoside analogues that are optionally included within oligomers mayfunction similarly to corresponding nucleosides, or may have specificimproved functions. Oligomers wherein some or all of the monomers arenucleoside analogues are often preferred over native forms because ofseveral desirable properties of such oligomers, such as the ability topenetrate a cell membrane, good resistance to extra- and/orintracellular nucleases and high affinity and specificity for thenucleic acid target. LNA monomers are particularly preferred, forexample, for conferring several of the above-mentioned properties.

In various embodiments, one or more nucleoside analogues present withinthe oligomer are “silent” or “equivalent” in function to thecorresponding natural nucleoside, i.e., have no functional effect on theway the oligomer functions to inhibit target gene expression. Such“equivalent” nucleoside analogues are nevertheless useful if, forexample, they are easier or cheaper to manufacture, or are more stableunder storage or manufacturing conditions, or can incorporate a tag orlabel. Typically, however, the analogues will have a functional effecton the way in which the oligomer functions to inhibit expression; forexample, by producing increased binding affinity to the target region ofthe target nucleic acid and/or increased resistance to intracellularnucleases and/or increased ease of transport into the cell.

Thus, in various embodiments, oligomers according to the inventioncomprise nucleoside monomers and at least one nucleoside analoguemonomer, such as an LNA monomer, or other nucleoside analogue monomers.

The term “at least one” comprises the integers larger than or equal to1, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 and so forth. In various embodiments, such as when referringto the nucleic acid or protein targets of the compounds of theinvention, the term “at least one” includes the terms “at least two” and“at least three” and “at least four.” Likewise, in some embodiments, theterm “at least two” comprises the terms “at least three” and “at leastfour.”

In some embodiments, the oligomer comprises or consists of 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30contiguous monomers.

In some embodiments, the oligomer comprises or consists of 10-22contiguous monomers, such as 12-18 contiguous monomers, such as 13-17 or12-16 contiguous monomers, such as 13, 14, 15, 16 contiguous monomers.

In certain embodiments, the oligomer comprises or consists of 10, 11,12, 13, or 14 contiguous monomers.

In various embodiments, the oligomer according to the invention consistsof no more than 22 monomers, such as no more than 20 monomers, such asno more than 18 monomers, such as 15, 16 or 17 monomers. In someembodiments, the oligomer of the invention comprises less than 20monomers.

In various embodiments, the compounds of the invention do not compriseRNA monomers.

In various embodiments, the compounds according to the invention arelinear molecules or are linear as synthesised. The oligomer, in suchembodiments, is a single stranded molecule, and typically does notcomprise short regions of, for example, at least 3, 4 or 5 contiguousmonomers, which are complementary to another region within the sameoligomer such that the oligomer forms an internal duplex. In someembodiments, the oligomer is essentially not double stranded, i.e., isnot a siRNA.

In some embodiments, the oligomer of the invention consists of acontiguous stretch of monomers, the sequence of which is identified by aSEQ ID NO disclosed herein (see, e.g., Tables 1-3). In otherembodiments, the oligomer comprises a first region, the regionconsisting of a contiguous stretch of monomers, and one or moreadditional regions which consist of at least one additional monomer. Insome embodiments, the sequence of the first region is identified by aSEQ ID NO disclosed herein.

Gapmer Design

Typically, the oligomer of the invention is a gapmer.

A “gapmer” is an oligomer which comprises a contiguous stretch ofmonomers capable of recruiting an RNAse (e.g., such as RNAseH) asfurther described herein below, such as a region of at least 6 or 7 DNAmonomers, referred to herein as region B, wherein region B is flankedboth on its 5′ and 3′ ends by regions respectively referred to asregions A and C, each of regions A and C comprising or consisting ofnucleoside analogues, such as affinity-enhancing nucleoside analogues,such as 1-6 nucleoside analogues.

Typically, the gapmer comprises regions, from 5′ to 3′, A-B-C, oroptionally A-BC-D or D-A-B-C, wherein: region A consists of or comprisesat least one nucleoside analogue, such as at least one LNA monomer, suchas 1-6 nucleoside analogues, such as LNA monomers, and region B consistsof or comprises at least five contiguous monomers which are capable ofrecruiting RNAse (when formed in a duplex with a complementary targetregion of the target RNA molecule, such as the mRNA target), such as DNAmonomers; region C consists of or comprises at least one nucleosideanalogue, such as at least one LNA monomer, such as 1-6 nucleosideanalogues, such as LNA monomers; and region D, when present, consists ofor comprises 1, 2 or 3 monomers, such as DNA monomers.

In various embodiments, region A consists of 1, 2, 3, 4, 5 or 6nucleoside analogues, such as LNA monomers, such as 2-5 nucleosideanalogues, such as 2-5 LNA monomers, such as 3 or 4 nucleosideanalogues, such as 3 or 4 LNA monomers, and/or region C consists of 1,2, 3, 4, 5 or 6 nucleoside analogues, such as LNA monomers, such as 2-5nucleoside analogues, such as 2-5 LNA monomers, such as 3 or 4nucleoside analogues, such as 3 or 4 LNA monomers.

In certain embodiments, region B consists of or comprises 5, 6, 7, 8, 9,10, 11 or 12 contiguous monomers which are capable of recruiting RNAse,or 6-10, or 7-9, such as 8 contiguous monomers which are capable ofrecruiting RNAse. In certain embodiments, region B consists of orcomprises at least one DNA monomer, such as 1-12 DNA monomers,preferably 4-12 DNA monomers, more preferably 6-10 DNA monomers, such as7-10 DNA monomers, most preferably 8, 9 or 10 DNA monomers.

In various embodiments, region A consists of 3 or 4 nucleosideanalogues, such as LNA monomers, region B consists of 7, 8, 9 or 10 DNAmonomers, and region C consists of 3 or 4 nucleoside analogues, such asLNA monomers. Such designs include (A-B-C) 3-10-3, 3-10-4, 4-10-3,3-9-3, 3-9-4, 4-9-3, 3-8-3, 3-84, 4-8-3, 3-7-3, 3-7-4, 4-7-3, and mayfurther include region D, which may have one or 2 monomers, such as DNAmonomers.

Further gapmer designs are disclosed in WO2004/046160, which is herebyincorporated by reference.

US provisional application, 60/977,409, hereby incorporated byreference, refers to ‘shortmer’ gapmer oligomers. In some embodiments,oligomers presented here may be such shortmer gapmers.

In certain embodiments, the oligomer consists of 10, 11, 12, 13 or 14contiguous monomers, wherein the regions of the oligomer have thepattern (5′-3′), A-B-C, or optionally A-B-C-D or D-A-B-C, wherein:region A consists of 1, 2 or 3 nucleoside analogue monomers, such as LNAmonomers; region B consists of 7, 8 or 9 contiguous monomers which arecapable of recruiting RNAse when formed in a duplex with a complementaryRNA molecule (such as a mRNA target); and region C consists of 1, 2 or 3nucleoside analogue monomers, such as LNA monomers. When present, regionD consists of a single DNA monomer.

In certain embodiments, region A consists of 1 LNA monomer. In certainembodiments, region A consists of 2 LNA monomers. In certainembodiments, region A consists of 3 LNA monomers. In certainembodiments, region C consists of 1 LNA monomer. In certain embodiments,region C consists of 2 LNA monomers. In certain embodiments, region Cconsists of 3 LNA monomers. In certain embodiments, region B consists of7 nucleoside monomers. In certain embodiments, region B consists of 8nucleoside monomers. In certain embodiments, region B consists of 9nucleoside monomers. In certain embodiments, region B comprises 1-9 DNAmonomers, such as 2, 3, 4, 5, 6, 7 or 8 DNA monomers. In certainembodiments, region B consists of DNA monomers. In certain embodiments,region B comprises at least one LNA monomer which is in the alpha-Lconfiguration, such as 2, 3, 4, 5, 6, 7, 8 or 9 LNA monomers in thealpha-L-configuration. In certain embodiments, region B comprises atleast one alpha-L-oxy LNA monomer. In certain embodiments, all the LNAmonomers in region B that are in the alpha-L configuration, arealpha-L-oxy LNA units. In certain embodiments, the number of monomerspresent in the A-B-C regions are selected from the group consisting of(nucleoside analogue monomers—region B—nucleoside analogue monomers):1-8-1, 1-8-2, 2-8-1, 2-8-2, 3-8-3, 2-8-3, 3-8-2, 4-8-1, 4-8-2, 1-8-4,2-8-4, or; 1-9-1, 1-9-2, 2-9-1, 2-9-2, 2-9-3, 3-9-2, 1-9-3, 3-9-1,4-9-1, 1-94, or; 1-10-1, 1-10-2, 2-10-1, 2-10-2, 1-10-3, 3-10-1. Incertain embodiments, the number of monomers present in the A-B-C regionsof the oligomer of the invention is selected from the group consistingof: 2-7-1, 1-7-2, 2-7-2, 3-7-3, 2-7-3, 3-7-2, 3-7-4, and 4-7-3. Incertain embodiments, each of regions A and C consists of two LNAmonomers, and region B consists of 8 or 9 nucleoside monomers,preferably DNA monomers.

In various embodiments, other gapmer designs include those where regionsA and/or C consists of 3, 4, 5 or 6 nucleoside analogues, such asmonomers containing a 2′-O-methoxyethyl-ribose sugar (2′-MOE) ormonomers containing a 2′-fluoro-deoxyribose sugar, and region B consistsof 8, 9, 10, 11 or 12 nucleosides, such as DNA monomers, where regionsA-B-C have 5-10-5 or 4-12-4 monomers. Further gapmer designs aredisclosed in WO 2007/146511A2, hereby incorporated by reference.

Internucleoside Linkages

The monomers of the oligomers described herein are coupled together vialinkage groups. Suitably, each monomer is linked to the 3′ adjacentmonomer via a linkage group.

The terms “linkage group” or “internucleoside linkage” means a groupcapable of covalently coupling together two contiguous monomers.Specific and preferred examples include phosphate groups (forming aphosphodiester between adjacent nucleoside monomers) andphosphorothioate groups (forming a phosphorothioate linkage betweenadjacent nucleoside monomers).

Suitable linkage groups include those listed in PCT/DK2006/000512, forexample in the first paragraph of page 34 of PCT/DK2006/000512 (herebyincorporated by reference).

It is, in various embodiments, preferred to modify the linkage groupfrom its normal phosphodiester to one that is more resistant to nucleaseattack, such as phosphorothioate or boranophosphate—these two beingcleavable by RNase H, thereby permitting RNase-mediated antisenseinhibition of expression of the target gene.

In some embodiments, suitable sulphur (S) containing linkage groups asprovided herein are preferred. In various embodiments, phosphorothioatelinkage groups are preferred, particularly for the gap region (B) ofgapmers. In certain embodiments, phosphorothioate linkages are used tolink together monomers in the flanking regions (A and C). In variousembodiments, phosphorothioate linkages are used for linking regions A orC to region D, and for linking together monomers within region D.

In various embodiments, regions A, B and C, comprise linkage groupsother than phosphorothioate, such as phosphodiester linkages,particularly, for instance when the use of nucleoside analogues protectsthe linkage groups within regions A and C from endo-nucleasedegradation—such as when regions A and C comprise LNA monomers.

In various embodiments, adjacent monomers of the oligomer are linked toeach other by means of phosphorothioate groups.

It is recognised that the inclusion of phosphodiester linkages, such asone or two linkages, into an oligomer with a phosphorothioate backbone,particularly with phosphorothioate linkage groups between or adjacent tonucleoside analogue monomers (typically in region A and/or C), canmodify the bioavailability and/or bio-distribution of an oligomer—seeWO2008/053314, hereby incorporated by reference.

In some embodiments, such as the embodiments referred to above, wheresuitable and not specifically indicated, all remaining linkage groupsare either phosphodiester or phosphorothioate, or a mixture thereof.

In some embodiments all the internucleoside linkage groups arephosphorothioate.

When referring to specific gapmer oligonucleotide sequences, such asthose provided herein, it will be understood that, in variousembodiments, when the linkages are phosphorothioate linkages,alternative linkages, such as those disclosed herein may be used, forexample phosphate phosphodiester) linkages may be used, particularly forlinkages between nucleoside analogues, such as LNA monomers. Likewise,in various embodiments, when referring to specific gapmeroligonucleotide sequences, such as those provided herein, when one ormore monomers in region C comprises a 5-methylcytosine base, othermonomers in that region may contain unmodified cytosine bases.

Target Nucleic Acid

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein, and are defined as a molecule formed by covalent linkage of twoor more monomers, as above-described. Including 2 or more monomers,“nucleic acids” may be of any length, and the term is generic to“oligomers”, which have the lengths described herein. The terms “nucleicacid” and “polynucleotide” include single-stranded, double-stranded,partially double-stranded, and circular molecules.

The term “target nucleic acid”, as used herein, refers to DNA or RNA(e.g., mRNA or pre-mRNA) encoding a mammalian androgen receptorpolypeptide, such as human androgen receptor, such as the nucleic acidhaving the sequence shown in SEQ ID NO: 1, and naturally occurringallelic variants of such nucleic acids. In certain embodiments, themammalian androgen receptor is a mouse androgen receptor. In someembodiments, for example when used in research or diagnostics, the“target nucleic acid” is a cDNA or a synthetic oligonucleotide derivedfrom the above DNA or RNA nucleic acid targets. The oligomers accordingto the invention are typically capable of hybridising to the targetnucleic acid.

Exemplary target nucleic acids include mammalian androgenreceptor-encoding nucleic acids having the GenBank Accession numbersshown in the table below, along with their corresponding proteinsequences:

GenBank Accession Number Nucleic acid (mRNA/cDNA GenBank AccessionNumber sequence) Polypeptide (deduced) Human NM_000044 NP_000035 MouseNM_013476 NP_038504 Rhesus NM_001032911 NP_001028083 monkey

It is recognised that the above-disclosed GenBank Accession numbers fornucleic acids refer to cDNA sequences and not to mRNA sequences per se.The sequence of a mature mRNA can be derived directly from thecorresponding cDNA sequence with thymine bases (T) being replaced byuracil bases (U).

The term “naturally occurring variant thereof” refers to variants of theandrogen receptor polypeptide or nucleic acid sequence which existnaturally within the defined taxonomic group, such as mammalian, such asmouse, monkey, and preferably human AR. Typically, when referring to“naturally occurring variants” of a polynucleotide the term alsoencompasses any allelic variant of the androgen receptor encodinggenomic DNA which is found at the Chromosome X: 66.68-66.87 Mb bychromosomal translocation or duplication, and the RNA, such as mRNAderived therefrom. “Naturally occurring variants” may also includevariants derived from alternative splicing of the androgen receptormRNA. When referenced to a specific polypeptide sequence, e.g., the termalso includes naturally occurring forms of the protein which maytherefore be processed, e.g. by co- or post-translational modifications,such as signal peptide cleavage, proteolytic cleavage, glycosylation,etc.

It is recognised that the human androgen receptor gene exhibits allelicvariations that are associated with disease phenotypes (Mooney et al,NAR 15; 31(8) 2003). For example, a (CAG)_(n) repeat expansion isassociated with polyglutamine expansion disorder. Other characterisedallelic variants include a (GOC)_(a) trinucleotide repeat and singlenucleotide polymorphisms R726L, T887A and L710H, of which the latter twosingle nucleotide polymorphisms have been shown to be correlated toenhanced promiscuity of the AR receptor for other steroid ligands. Inone embodiment “n” ranges from 5-31. CAG repeats of less than 22 havebeen associated with an enhanced risk of prostate cancer in AfricanAmerican males.

In various embodiments, the target nucleic acid is an AR allelic variantwhich comprises a (CAG)_(n) trinucleotide repeat, or (GGC)_(n)trinucleotide repeat. In other embodiments, the target nucleic acid isan AR allelic variant which comprises one or more single nucleotidepolymorphisms, including R726L, T887A and L710H.

In certain embodiments, oligomers described herein bind to a region ofthe target nucleic acid (the “target region”) by either Watson-Crickbase pairing, Hoogsteen hydrogen bonding, or reversed Hoogsteen hydrogenbonding, between the monomers of the oligomer and monomers of the targetnucleic acid. Such binding is also referred to as “hybridisation.”Unless otherwise indicated, binding is by Watson-Crick pairing ofcomplementary bases (i.e., adenine with thymine (DNA) or uracil (RNA),and guanine with cytosine), and the oligomer binds to the target regionbecause the sequence of the oligomer is identical to, orpartially-identical to, the sequence of the reverse complement of thetarget region; for purposes herein, the oligomer is said to be“complementary” or “partially complementary” to the target region, andthe percentage of “complementarity” of the oligomer sequence to that ofthe target region is the percentage “identity” to the reverse complementof the sequence of the target region.

Unless otherwise made clear by context, the “target region” herein willbe the region of the target nucleic acid having the sequence that bestaligns with the reverse complement of the sequence of the specifiedoligomer (or region thereof), using the alignment program and parametersdescribed herein below.

In determining the degree of “complementarity” between oligomers of theinvention (or regions thereof) and the target region of the nucleic acidwhich encodes mammalian androgen receptor, such as those disclosedherein, the degree of “complementarity” (also, “homology”) is expressedas the percentage identity between the sequence of the oligomer (orregion thereof) and the reverse complement of the sequence of the targetregion that best aligns therewith. The percentage is calculated bycounting the number of aligned bases that are identical as between the 2sequences, dividing by the total number of contiguous monomers in theoligomer, and multiplying by 100. In such a comparison, if gaps exist,it is preferable that such gaps are merely mismatches rather than areaswhere the number of monomers within the gap differs between the oligomerof the invention and the target region.

Amino acid and polynucleotide alignments, percentage sequence identity,and degree of complementarity may be determined for purposes of theinvention using the ClustalW algorithm using standard settings: seehttp://www.ebi.ac.uk/emboss/align/index.html, Method: EMBOSS::water(local): Gap Open=10.0, Gap extend=0.5, using Blosum 62 (protein), orDNAfull for nucleotide/nucleobase sequences.

As will be understood, depending on context, “mismatch” refers to anon-identity in sequence (as, for example, between the nucleobasesequence of an oligomer and the reverse complement of the target regionto which it binds; as for example, between the base sequence of twoaligned AR encoding nucleic acids), or to noncomplementarity in sequence(as, for example, between an oligomer and the target region to which itbinds).

The androgen receptor is known to regulate the expression of severalgenes, such as a gene selected from the group consisting of Proteinkinase C delta (PRKCD), Glutathione S-transferase theta 2 (GSTT2),transient receptor potential cation channel subfamily V member 3(TRPV3), Pyrroline-5-carboxylate reductase 1 (PYCR1) and ornithineaminotransferase (OAT). Such genes regulated by AR are referred toherein as “androgen receptor (AR) target genes”. In various embodiments,the oligomers according to the invention are capable of inhibiting (suchas, by down-regulating) the expression of one or more AR target genes ina cell which is expressing, or is capable of expressing (i.e. byalleviating AR repression of the AR target gene in a cell) an AR targetgene.

The oligomers which target the androgen receptor mRNA, may hybridize toany site along the target mRNA nucleic acid, such as the 5′ untranslatedleader, exons, introns and 3′ untranslated tail. However, it ispreferred that the oligomers which target the androgen receptor mRNAhybridise to the mature mRNA form of the target nucleic acid.

Suitably, the oligomer of the invention or conjugate thereof is capableof down-regulating expression of the androgen receptor gene. In variousembodiments, the oligomer (or conjugate) of the invention can effect theinhibition of androgen receptor, typically in a mammalian cell, such asa human cell. In certain embodiments, the oligomers of the invention, orconjugates thereof, bind to the target nucleic acid and effectinhibition of AR mRNA expression of at least 10% or 20% compared to theexpression level immediately prior to dosing of the oligomer, morepreferably of at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% ascompared to the AR expression level immediately prior to dosing of theoligomer. In some embodiments, such inhibition is seen when using fromabout 0.04 nM to about 25 nM, such as from about 0.8 nM to about 20 nMof the oligomer or conjugate.

In various embodiments, the inhibition of mRNA expression is less than100% (i.e., less than complete inhibition of expression), such as lessthan 98% inhibition, less than 95% inhibition, less than 90% inhibition,less than 80% inhibition, such as less than 70% inhibition. In variousembodiments, modulation of gene expression can be determined bymeasuring protein levels, e.g. by the methods such as SDS-PAGE followedby western blotting using suitable antibodies raised against the targetprotein. Alternatively, modulation of expression levels can bedetermined by measuring levels of mRNA, e.g. by northern blotting orquantitative RT-PCR. When measuring via mRNA levels, the level ofdown-regulation when using an appropriate dosage, such as from about0.04 nM to about 25 nM, such as from about 0.8 nM to about 20 nM, is, invarious embodiments, typically to a level of 10-20% of the normal levelsin the absence of the compound or conjugate of the invention.

The invention therefore provides a method of down-regulating orinhibiting the expression of the androgen receptor protein and/or mRNAin a cell which is expressing the androgen receptor protein and/or mRNA,the method comprising contacting the cell with an effective amount ofthe oligomer or conjugate according to the invention to down-regulate orinhibit the expression of the androgen receptor protein and/or mRNA inthe cell. Suitably the cell is a mammalian cell, such as a human cell.The contacting may occur, in some embodiments, in vitro. The contactingmay occur, in some embodiments, in vivo.

Oligomer Sequences

In some embodiments, the oligomers of the invention have sequences thatare identical to a sequence selected from the group consisting of SEQ IDNOS: 2-22. Target regions in human AR mRNA (cDNA) that bind to theoligomers having sequences as set forth in SEQ ID NOs: 2-22 are shown inFIG. 4 (bold and underlined, with the corresponding oligomer SEQ ID NOsindicated above).

Further provided are target nucleic acids (e.g., DNA or mRNA encodingAR) that contain target regions that are complementary orpartially-complementary to one or more of the oligomers of theinvention. In certain embodiments, the oligomers bind to variants of ARtarget regions, such as allelic variants (such as an AR gene present atgene locus Xq11-12). In some embodiments, a variant of an AR targetregion has at least 60%, more preferably at least 70%, more preferablyat least 80%, more preferably at least 85%, more preferably at least90%, more preferably at least 91%, at least 92%, at least 93%, at least94%, at least 95% sequence identity to the target region in wild-typeAR. Thus, in other embodiments, the oligomers of the invention havesequences that differ in 1, 2 or 3 bases when compared to a sequenceselected from the group consisting of SEQ ID NOs: 2-22. Typically, anoligomer of the invention that binds to a variant of an AR target regionis capable of inhibiting (e.g., by down-regulating) AR.

In other embodiments, oligomers of the invention are LNA oligomers, forexample, those oligomers having the sequences shown in SEQ ID NOs:44-80. In various embodiments, the oligomers of the invention are potentinhibitors of androgen receptor mRNA and protein expression. In variousembodiments, oligomers of the invention are LNA oligomers having thesequences of SEQ ID NO: 58 or SEQ ID NO: 77.

In various embodiments, the oligomer comprises or consists of a regionhaving a base sequence which is identical or partially identical to thesequence of the reverse complement of a target region in SEQ ID NO: 1.In various embodiments, the oligomer comprises or consists of a regionhaving a sequence selected from the group consisting of SEQ ID NOS: 2-22and 86-106.

In certain embodiments, the oligomer comprises or consists of a regionhaving a base sequence which is fully complementary (perfectlycomplementary) to a target region of a nucleic acid which encodes amammalian androgen receptor.

However, in some embodiments, the oligomer includes 1, 2, 3, or 4 (ormore) mismatches as compared to the best-aligned target region of an ARtarget nucleic acid, and still sufficiently binds to the target regionto effect inhibition of AR mRNA or protein expression. The destabilizingeffect of mismatches on Watson-Crick hydrogen-bonded duplex may, forexample, be compensated by increased length of the oligomer and/or anincreased number of nucleoside analogues, such as LNA monomers, presentwithin the oligomer.

In various embodiments, the oligomer base sequence comprises no morethan 3, such as no more than 2 mismatches compared to the base sequenceof the best-aligned target region of, for example, a target nucleic acidwhich encodes a mammalian androgen receptor.

In some embodiments, the oligomer base sequence comprises no more than asingle mismatch when compared to the base sequence of the best-alignedtarget region of a nucleic acid which encodes a mammalian androgenreceptor.

In various embodiments, the base sequence of the oligomer of theinvention, or of a first region thereof, is preferably at least 80%identical to a base sequence selected from the group consisting of SEQID NOS: 2-22 and 86-106, such as at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96% identical, such as 100% identical.

In certain embodiments, the base sequence of the oligomer of theinvention or of a first region thereof is at least 80% identical to thebase sequence of the reverse complement of a target region present inSEQ ID NO: 1, such as at least 85%, at least 90%, at least 91%, at least92% at least 93%, at least 94%, at least 95%, at least 96% identical, atleast 97% identical, at least 98% identical, at least 99% identical,such as 100% identical.

In various embodiments, the base sequence of the oligomer of theinvention, or of a first region thereof, is preferably at least 80%complementary to a target region of SEQ ID NO: 1, such as at least 85%,at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96% complementary, at least 97% complementary, atleast 98% complementary, at least 99% complementary, such as 100%complementary (perfectly complementary).

In some embodiments the oligomer (or a first region thereof) has a basesequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22, oris selected from the group consisting of at least 10 contiguous monomersof SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, and 22. In other embodiments, the sequence of theoligomer of the invention or a first region thereof comprises one, two,or three base moieties that differ from those in oligomers havingsequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, or 22, or the sequences of at least 10contiguous monomers thereof, when optimally aligned with the selectedsequence or region thereof.

In some embodiments the oligomer (or a first region thereof) has a basesequence selected from the group consisting of SEQ ID NOs: 86, 87, 88,89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105and 106, or the sequences of at least 10 contiguous monomers thereof. Inother embodiments, the sequence of the oligomer (or a first regionthereof) comprises one, two, or three base moieties that differ fromthose in oligomers having sequences of SEQ ID NOs: 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 or 106,or the sequences of at least 10 contiguous monomers thereof, whenoptimally aligned with the selected sequence or region thereof.

In various embodiments, the oligomers comprise a region of 12, 13, 14,15 or 16 contiguous monomers having a base sequence identically presentin a sequence selected from the group consisting of SEQ ID No 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and 22.In other embodiments, the oligomers include a region which comprisesone, two, or three base moieties that differ from those in oligomershaving sequences of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, or 22.

In some embodiments the region consists of 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 contiguous monomers,such as 12-22, such as 12-18 monomers. Suitably, in some embodiments,the region is of the same length as the oligomer of the invention.

In some embodiments the oligomer comprises additional monomers at the 5′or 3′ ends, such as, independently, 1, 2, 3, 4 or 5 additional monomersat the 5′ end and/or the 3′ end of the oligomer, which arenon-complementary to the target region. In various embodiments, theoligomer of the invention comprises a region that is complementary tothe target, which is flanked 5′ and/or 3′ by additional monomers. Insome embodiments the additional 5′ or 3′ monomers are nucleosides, suchas DNA or RNA monomers. In various embodiments, the 5′ or 3′ monomersrepresent region D as referred to in the context of gapmer oligomersherein.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO:2, such as SEQ ID NO: 44, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID No: 3, such as SEQ ID NO: 45, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 4, such as SEQ ID NO: 46, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 5, such as SEQ ID NO: 47, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 6, such as SEQ ID NOs: 48, 49 or 50, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 7, such as SEQ ID NOs: 51, 52, or 53, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 8, such as SEQ ID NOs: 54, 55 or 56, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 9, such as SEQ ID NO: 57, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 10, such as SEQ ID NOs: 58, 59, or 60, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 11, such as SEQ ID NO: 61, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 12, such as SEQ ID NO: 62, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 13, such as SEQ ID NOs: 63, 64 or 65, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 14, such as SEQ ID NO: 66, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 15, such as SEQ ID NO: 67, or according to aregion of at least 10 contiguous monomers thereof such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 16, such as SEQ ID NO: 68, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 17, such as SEQ ID NOs: 69, 70 or 71, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 18, such as SEQ ID NO: 72, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 19, such as SEQ ID NOs: 73, 74 or 75, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 20, such as SEQ ID NO: 76, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 21, such as SEQ ID NOs: 77, 78 or 79, oraccording to a region of at least 10 contiguous monomers thereof, suchas 11, 12, 13, 14, 15 or 16 contiguous monomers thereof.

In certain embodiments, the oligomer according to the invention consistsof or comprises contiguous monomers having a nucleobase sequenceaccording to SEQ ID NO: 22, such as SEQ ID NO: 80, or according to aregion of at least 10 contiguous monomers thereof, such as 11, 12, 13,14, 15 or 16 contiguous monomers thereof.

Nucleosides and Nucleoside Analogues

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified base, such asa base selected from 5-methylcytosine, isocytosine, pseudoisocytosine,5-bromouracil, 5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine,diaminopurine, 2-chloro-6-aminopurine, xanthine and hypoxanthine.

In various embodiments, at least one of the monomers present in theoligomer is a nucleoside analogue that contains a modified sugar.

In some embodiments, the linkage between at least 2 contiguous monomersof the oligomer is other than a phosphodiester linkage.

In certain embodiments, the oligomer includes at least one monomer thathas a modified base, at least one monomer (which may be the samemonomer) that has a modified sugar, and at least one inter-monomerlinkage that is non-naturally occurring.

Specific examples of nucleoside analogues are described by e.g. Freier &Altmann; Nucl. Acid Res., 1997, 25, 44294443 and Uhlmann; Curr. Opinionin Drug Development, 2000, 3(2), 293-213, and in Scheme 1 (in which somenucleoside analogues are shown as nucleotides):

The oligomer may thus comprise or consist of a simple sequence ofnaturally occurring nucleosides—preferably DNA monomers, but alsopossibly RNA monomers, or a combination of nucleosides and one or morenucleoside analogues. In some embodiments, such nucleoside analoguessuitably enhance the affinity of the oligomer for the target region ofthe target nucleic acid.

Examples of suitable and preferred nucleoside analogues are described inPCT/DK2006/000512, or are referenced therein.

In some embodiments, the nucleoside analogue comprises a sugar moietymodified to provide a 2′-substituent group, such as 2′-O-alkyl-ribosesugars, 2′-amino-deoxyribose sugars, and 2′-fluoro-deoxyribose sugars.

In some embodiments, the nucleoside analogue comprises a sugar in whicha bridged structure, creating a bicyclic sugar (LNA), which enhancesbinding affinity and may also provide some increased nucleaseresistance. In various embodiments, the LNA monomer is selected fromoxy-LNA (such as beta-D-oxy-LNA, and alpha-L-oxy-LNA), and/or amino-LNA(such as beta-D-amino-LNA and alpha-L-amino-LNA) and/or thio-LNA (suchas beta-D-thio-LNA and alpha-L-thio-LNA) and/or ENA (such as beta-D-ENAand alpha-L-ENA). In certain embodiments, the LNA monomers arebeta-D-oxy-LNA. LNA monomers are further described below.

In various embodiments, incorporation of affinity-enhancing nucleosideanalogues in the oligomer, such as LNA monomers or monomers containing2′-substituted sugars, or incorporation of modified linkage groupsprovides increased nuclease resistance. In various embodiments,incorporation of affinity-enhancing nucleoside analogues allows the sizeof the oligomer to be reduced, and also reduces the size of the oligomerthat binds specifically to a target region of a target sequence.

In some embodiments, the oligomer comprises at least 2 nucleosideanalogues. In some embodiments, the oligomer comprises from 3-8nucleoside analogues, e.g. 6 or 7 nucleoside analogues. In variousembodiments, at least one of the nucleoside analogues is a lockednucleic acid (LNA) monomer; for example at least 3 or at least 4, or atleast 5, or at least 6, or at least 7, or 8, nucleoside analogues areLNA monomers. In some embodiments, all the nucleoside analogues are LNAmonomers.

It will be recognised that when referring to a preferred oligomer basesequence, in certain embodiments, the oligomers comprise a correspondingnucleoside analogue, such as a corresponding LNA monomer or othercorresponding nucleoside analogue, which raise the duplex stability(T_(m)) of the oligomer/target region duplex (i.e. affinity enhancingnucleoside analogues).

In various embodiments, any mismatches (i.e., non-complementarities)between the base sequence of the oligomer and the base sequence of thetarget region, if present, are preferably located other than in theregions of the oligomer that contain affinity-enhancing nucleosideanalogues (e.g., regions A or C), such as within region B as referred toherein, and/or within region D as referred to herein, and/or in regionsconsisting of DNA monomers, and/or in regions which are 5′ or 3′ to theregion of the oligomer that is complementary to the target region.

In some embodiments the nucleoside analogues present within the oligomerof the invention (such as in regions A and C mentioned herein) areindependently selected from, for example: monomers containing2′-O-alkyl-ribose sugars, monomers containing 2′-amino-deoxyribosesugars, monomers containing 2′-fluoro-deoxyribose sugars, LNA monomers,monomers containing arabinose sugars (“ANA monomers”), monomerscontaining 2′-fluoroarabinose sugars, monomers containingd-arabino-hexitol sugars (“HNA monomers”), intercalating monomers asdefined in Christensen (2002) Nucl. Acids. Res. 30: 4918-4925, herebyincorporated by reference, and 2′-O-methoxyethyl-ribose (2′MOE) sugars.In some embodiments, there is only one of the above types of nucleosideanalogues present in the oligomer of the invention, or region thereof.

In certain embodiments, the nucleoside analogues contain 2′MOE sugars,2′-fluoro-deoxyribose sugars, or LNA sugars, and as such theoligonucleotide of the invention may comprise nucleoside analogues whichare independently selected from these three types. In certain oligomerembodiments containing nucleoside analogues, at least one of saidnucleoside analogues contains a 2′-MOE-ribose sugar, such as 2, 3, 4, 5,6, 7, 8, 9 or 10 nucleoside analogues containing 2′-MOE-ribose sugars.In some embodiments, at least one nucleoside analogue contains a2′-fluoro-deoxyribose sugar, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10nucleoside analogues containing 2′-fluoro-DNA nucleotide sugars.

In various embodiments, the oligomer according to the inventioncomprises at least one Locked Nucleic Acid (LNA) monomer, such as 1, 2,3, 4, 5, 6, 7, or 8 LNA monomers, such as 3-7 or 4 to 8 LNA monomers, or3, 4, 5, 6 or 7 LNA monomers. In various embodiments, all the nucleosideanalogues are LNA monomers. In certain embodiments, the oligomercomprises both beta-D-oxy-LNA monomers, and one or more of the followingLNA monomers: thio-LNA monomers, amino-LNA monomers, oxy-LNA monomers,and/or ENA monomers in either the beta-D or alpha-L configurations, orcombinations thereof. In certain embodiments, the cytosine base moietiesof all LNA monomers in the oligomer are 5-methylcytosines. In certainembodiments of the invention, the oligomer comprises both LNA and DNAmonomers. Typically, the combined total of LNA and DNA monomers is10-25, preferably 10-20, even more preferably 12-16. In some embodimentsof the invention, the oligomer or region thereof consists of at leastone LNA monomer, and the remaining monomers are DNA monomers. In certainembodiments, the oligomer comprises only LNA monomers and nucleosides(such as RNA or DNA monomers, most preferably DNA monomers) optionallywith modified linkage groups such as phosphorothioate.

In various embodiments, at least one of the nucleoside analogues presentin the oligomer has a modified base selected from the group consistingof 5-methylcytosine, isocytosine, pseudoisocytosine, 5-bromouracil,5-propynyluracil, 6-aminopurine, 2-aminopurine, inosine, diaminopurine,and 2-chloro-6-aminopurine.

LNA

The term “LNA monomer” refers to a nucleoside analogue containing abicyclic sugar (an “LNA sugar”). The terms “LNA oligonucleotide” and“LNA oligomer” refer to an oligomer containing one or more LNA monomers.

The LNA used in the oligonucleotide compounds of the inventionpreferably has the structure of the general formula I:

wherein X is selected from —O—, —S—, —N(R^(N)*)—, —C(R⁶R⁶*)—;

B is selected from hydrogen, optionally substituted C₁₋₄-alkoxy,optionally substituted C₁₋₄-alkyl, optionally substituted C₁₋₄-acyloxy,nucleobases, DNA intercalators, photochemically active groups,thermochemically active groups, chelating groups, reporter groups, andligands;

P designates the radical position for an internucleoside linkage to asucceeding monomer, or a 5′-terminal group, such internucleoside linkageor 5′-terminal group optionally including the substituent R⁵ or equallyapplicable the substituent R⁵*;

P* designates an internucleoside linkage to a preceding monomer, or a3′-terminal group;

R⁴* and R²* together designate a biradical consisting of 1-4groups/atoms selected from —C(R^(a)R^(b))—, —C(R^(a))═C(R^(b))—,—C(R^(a))═N—, —O—, —Si(R^(a))₂—, —S—, —SO₂—, —N(R^(a))—, and >C=Z,

wherein Z is selected from —O—, —S—, and —N(R^(a))—, and R^(a) and R^(b)each is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₁₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-aminocarbonyl, amino-C₁₋₄-alkyl-aminocarbonyl, mono anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),and

each of the substituents R¹, R², R³, R⁵, R⁵*, R⁶ and R⁶*, which arepresent is independently selected from hydrogen, optionally substitutedC₁₋₁₂-alkyl, optionally substituted C₂₋₁₂-alkenyl, optionallysubstituted C₂₋₂-alkynyl, hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl,C₂₋₁₂-alkenyloxy, carboxy, C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl,formyl, aryl, aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxycarbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-amino-carbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted, and where two geminal substituents together maydesignate oxo, thioxo, imino, or optionally substituted methylene, ortogether may form a spiro biradical consisting of a 1-5 carbon atom(s)alkylene chain which is optionally interrupted and/or terminated by oneor more heteroatoms/groups selected from —O—, —S—, and —(NR^(N))— whereR^(N) is selected from hydrogen and C₁₋₄-alkyl, and where two adjacent(non-geminal) substituents may designate an additional bond resulting ina double bond; and R^(N)*, when present and not involved in a biradical,is selected from hydrogen and C₁₋₄-alkyl; and basic salts and acidaddition salts thereof;

In some embodiments, R⁵* is selected from H, —CH₃, —CH₂—CH₃, —CH₂—O—CH₃,and —CH═CH₂.

In various embodiments, R⁴* and R²* together designate a biradicalselected from —C(R^(a)R^(b))—O—, —C(R^(a)R^(b))—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—O—,—C(R^(a)R^(b))—O—C(R^(c)R^(d))—, —C(R^(a)R^(b))—O—C(R^(c)R^(d))—O—,—C(R^(a)R^(b))—C(R^(c)R^(d))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—C(R^(e)R^(f))—,—C(R^(a))═C(R^(b))—C(R^(c)R^(d))—, —C(R^(a)R^(b))—N(R^(c))—,—C(R^(a)R^(b))—C(R^(c)R^(d))—N(R^(e))—, —C(R^(a)R^(b))—N(R^(c))—O—, and—C(R^(a)R^(b))—S—, —C(R^(a)R^(b))—C(R^(c)R^(d))—S—, wherein R^(a),R^(b), R^(c), R^(d), R^(e), and R^(f) each is independently selectedfrom hydrogen, optionally substituted C₁₋₁₂-alkyl, optionallysubstituted C₂₋₁₂-alkenyl, optionally substituted C₂₋₁₂-alkynyl,hydroxy, C₁₋₁₂-alkoxy, C₂₋₁₂-alkoxyalkyl, C₂₋₁₂-alkenyloxy, carboxy,C₁₋₁₂-alkoxycarbonyl, C₁₋₁₂-alkylcarbonyl, formyl, aryl,aryloxy-carbonyl, aryloxy, arylcarbonyl, heteroaryl,heteroaryloxy-carbonyl, heteroaryloxy, heteroarylcarbonyl, amino, mono-and di(C₁₋₆-alkyl)amino, carbamoyl, mono- anddi(C₁₋₆-alkyl)-aminocarbonyl, amino-C₁₋₆-alkyl-aminocarbonyl, mono- anddi(C₁₋₆-alkyl)amino-C₁₋₆-alkyl-aminocarbonyl, C₁₋₆-alkyl-carbonylamino,carbamido, C₁₋₆-alkanoyloxy, sulphono, C₁₋₆-alkylsulphonyloxy, nitro,azido, sulphanyl, C₁₋₆-alkylthio, halogen, DNA intercalators,photochemically active groups, thermochemically active groups, chelatinggroups, reporter groups, and ligands, where aryl and heteroaryl may beoptionally substituted and where two geminal substituents R^(a) andR^(b) together may designate optionally substituted methylene (═CH₂),

In a further embodiment R⁴* and R²* together designate a biradical(bivalent group) selected from —CH₂—O—, —CH₂—S—, —CH₂—NH—, —CH₂—N(CH₃)—,—CH₂—CH₂—O—, —CH₂—CH(CH₃)—, —CH₂—CH₂—S—, —CH₂—CH₂—NH—, —CH₂—CH₂—CH₂—,—CH₂—CH₂—CH₂—O—, —CH₂—CH₂—CH(CH₃)—, —CH═CH—CH₂—, —CH₂—O—CH₂—O—,—CH₂—NH—O—, —CH₂—N(CH₃)—O—, —CH₂—O—CH₂, —CH(CH₃)—O—, —CH(CH₂—O—CH₃)—O—.

For all chiral centers, asymmetric groups may be found in either R or Sorientation.

Preferably, the LNA monomer used in the oligomer of the inventioncomprises at least one LNA monomer according to any of the formulas

wherein Y is —O—, —O—CH₂—, —S—, —NH—, or N(RH); Z and Z* areindependently selected among an internucleotide linkage, a terminalgroup or a protecting group; B constitutes a natural or non-naturalnucleotide base moiety, and RH is selected from hydrogen and C₁₋₄-alkyl.

Specifically preferred LNA monomers are shown in Scheme 2:

The term “thio-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from S or CH₂—S—. Thio-LNA can be in eitherthe beta-D or alpha-L-configuration.

The term “amino-LNA” refers to an LNA monomer in which Y in the generalformula above is selected from —N(H)—, N(R)—, CH₂—N(H)—, and —CH₂—N(R)—where R is selected from hydrogen and C₁₋₄-alkyl. Amino-LNA can be ineither the beta-D or alpha-L-configuration.

The term “oxy-LNA” refers to an LNA monomer in which Y in the generalformula above represents —O— or —CH₂—O—. Oxy-LNA can be in either thebeta-D or alpha-L-configuration.

The term “ENA” refers to an LNA monomer in which Y in the generalformula above is —CH₂—O— (where the oxygen atom of —CH₂—O— is attachedto the 2′-position relative to the base B).

In various embodiments, the LNA monomer is selected from abeta-D-oxy-LNA monomer, and alpha-L-oxy-LNA monomer, a beta-D-amino-LNAmonomer, and beta-D-thio-LNA monomer, in particular a beta-D-oxy-LNAmonomer.

In the present context, the term “C₁₋₄ alkyl” means a linear or branchedsaturated hydrocarbon chain wherein the chain has from one to fourcarbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl,isobutyl, sec-butyl and tert-butyl.

RNAse H Recruitment

In some embodiments, an oligomer functions via non-RNase-mediateddegradation of a target mRNA, such as by steric hindrance oftranslation, or other mechanisms; however, in various embodiments,oligomers of the invention are capable of recruiting anendo-ribonuclease (RNase), such as RNase H.

Typically, the oligomer, comprises a region of at least 6, such as atleast 7 contiguous monomers, such as at least 8 or at least 9 contiguousmonomers, including 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 contiguousmonomers, which, when forming a duplex with the target region of thetarget RNA, is capable of recruiting RNase. The region of the oligomerwhich is capable of recruiting RNAse may be region B, as referred to inthe context of a gapmer as described herein. In some embodiments, theregion of the oligomer which is capable of recruiting RNAse, such asregion B, consists of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20monomers.

EP 1 222 309 provides in vitro methods for determining RNaseH activity,which may be used to determine the ability of the oligomers of theinvention to recruit RNaseH. An oligomer is deemed capable of recruitingRNaseH if, when contacted with the complementary region of the RNAtarget, it has an initial rate, as measured in pmol/l/min, of at least1%, such as at least 5%, such as at least 10% or less tan 20% of anoligonucleotide having the same base sequence but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Examples 91-95 of EP 1 222 309, incorporated herein byreference.

In some embodiments, an oligomer is deemed essentially incapable ofrecruiting RNaseH if, when contacted with the complementary targetregion of the RNA target, and RNaseH, the RNaseH initial rate, asmeasured in pmol/l/min, is less than 1%, such as less than 5%, such asless than 10% or less than 20% of the initial rate determined using anoligonucleotide having the same base sequence, but containing only DNAmonomers, with no 2′ substitutions, with phosphorothioate linkage groupsbetween all monomers in the oligonucleotide, using the methodologyprovided by Examples 91-95 of EP 1 222 309.

In other embodiments, an oligomer is deemed capable of recruiting RNaseHif, when contacted with the complementary target region of the RNAtarget, and RNaseH, the RNaseH initial rate, as measured in pmol/l/min,is at least 20%, such as at least 40%, such as at least 60%, such as atleast 80% of the initial rate determined using an oligonucleotide havingthe same base sequence, but containing only DNA monomers, with no 2′substitutions, with phosphorothioate linkage groups between all monomersin the oligonucleotide, using the methodology provided by Examples 91-95of EP 1 222 309.

Typically, the region of the oligomer which forms the duplex with thecomplementary target region of the target RNA and is capable ofrecruiting RNase contains DNA monomers and LNA monomers and forms aDNA/RNA-tike duplex with the target region. The LNA monomers arepreferably in the alpha-L configuration, particularly preferred beingalpha-L-oxy LNA.

In various embodiments, the oligomer of the invention comprises bothnucleosides and nucleoside analogues, and is in the form of a gapmer, aheadmer or a mixmer.

A “headmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch of non-RNase recruitingnucleoside analogues and the second region comprises a contiguousstretch (such as at least 7 contiguous monomers) of DNA monomers ornucleoside analogue monomers recognizable and cleavable by the RNase

A “tailmer” is defined as an oligomer that comprises a first region anda second region that is contiguous thereto, with the 5′-most monomer ofthe second region linked to the 3′-most monomer of the first region. Thefirst region comprises a contiguous stretch (such as at least 7contiguous monomers) of DNA monomers or nucleoside analogue monomersrecognizable and cleavable by the RNase, and the second region comprisesa contiguous stretch of non-RNase recruiting nucleoside analogues.

Other “chimeric” oligomers, called “mixmers”, consist of an alternatingcomposition of (i) DNA monomers or nucleoside analogue monomersrecognizable and cleavable by RNase, and (ii) non-RNase recruitingnucleoside analogue monomers.

In some embodiments, in addition to enhancing affinity of the oligomerfor the target region, some nucleoside analogues also mediate RNase(e.g., RNaseH) binding and cleavage. Since •-L-LNA monomers recruitRNaseH activity to a certain extent, in some embodiments, gap regions(e.g., region B as referred to herein) of oligomers containing •-L-LNAmonomers consist of fewer monomers recognizable and cleavable by theRNaseH, and more flexibility in the mixmer construction is introduced.

Conjugates

In the context of this disclosure, the term “conjugate” indicates acompound formed by the covalent attachment (“conjugation”) of anoligomer as described herein, to one or more moieties that are notthemselves nucleic acids or monomers (“conjugated moieties”). Examplesof such conjugated moieties include macromolecular compounds such asproteins, fatty acid chains, sugar residues, glycoproteins, polymers, orcombinations thereof. Typically proteins may be antibodies for a targetprotein. Typical polymers may be polyethylene glycol.

Accordingly, provided herein are conjugates comprising an oligomer asherein described, and at least one conjugated moiety that is not anucleic acid or monomer, covalently attached to said oligomer.Therefore, in certain embodiments where the oligomer of the inventionconsists of contiguous monomers having a specified sequence of bases, asherein disclosed, the conjugate may also comprise at least oneconjugated moiety that is covalently attached to the oligomer.

In various embodiments of the invention, the oligomer is conjugated to amoiety that increases the cellular uptake of oligomeric compounds.WO2007/031091 provides suitable ligands and conjugates, which are herebyincorporated by reference.

In various embodiments, conjugation (to a conjugated moiety) may enhancethe activity, cellular distribution or cellular uptake of the oligomerof the invention. Such moieties include, but are not limited to,antibodies, polypeptides, lipid moieties such as a cholesterol moiety,cholic acid, a thioether, e.g. Hexyl-s-tritylthiol, a thiocholesterol,an aliphatic chain, e.g., dodecandiol or undecyl residues, aphospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-o-hexadecyl-rac-glycero-3-h-phosphonate, a polyamine or apolyethylene glycol chain, an adamantane acetic acid, a palmityl moiety,an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.

In certain embodiments, the oligomers of the invention are conjugated toactive drug substances, for example, aspirin, ibuprofen, a sulfa drug,an antidiabetic, an antibacterial or an antibiotic.

In certain embodiments the conjugated moiety is a sterol, such ascholesterol.

In various embodiments, the conjugated moiety comprises or consists of apositively charged polymer, such as a positively charged peptides of,for example 1-50, such as 2-20 such as 3-10 amino acid residues inlength, and/or polyalkylene oxide such as polyethylene glycol (PEG) orpolypropylene glycol—see WO 2008/034123, hereby incorporated byreference. Suitably the positively charged polymer, such as apolyalkylene oxide may be attached to the oligomer of the invention viaa linker such as the releasable linker described in WO 2008/034123.

By way of example, the following moieties may be used in the conjugatesof the invention:

Activated Oligomers

The term “activated oligomer,” as used herein, refers to an oligomer ofthe invention that is covalently linked (i.e., functionalized) to atleast one functional moiety that permits covalent linkage of theoligomer to one or more conjugated moieties, i.e., moieties that are notthemselves nucleic acids or monomers, to form the conjugates hereindescribed. Typically, a functional moiety will comprise a chemical groupthat is capable of covalently bonding to the oligomer via, e.g., a3′-hydroxyl group or the exocyclic NH₂ group of the adenine base, aspacer that is preferably hydrophilic and a terminal group that iscapable of binding to a conjugated moiety (e.g., an amino, sulfhydryl orhydroxyl group). In some embodiments, this terminal group is notprotected, e.g., is an NH₂ group. In other embodiments, the terminalgroup is protected, for example, by any suitable protecting group suchas those described in “Protective Groups in Organic Synthesis” byTheodora W. Greene and Peter G. M. Wuts, 3rd edition (John Wiley & Sons,1999). Examples of suitable hydroxyl protecting groups include esterssuch as acetate ester, aralkyl groups such as benzyl, diphenylmethyl, ortriphenylmethyl, and tetrahydropyranyl. Examples of suitable aminoprotecting groups include benzyl, alpha-methylbenzyl, diphenylmethyl,triphenylmethyl, benzyloxycarbonyl, tert-butoxycarbonyl, and acyl groupssuch as trichloroacetyl or trifluoroacetyl.

In some embodiments, the functional moiety is self-leaving. In otherembodiments, the functional moiety is biodegradable. See e.g., U.S. Pat.No. 7,087,229, which is incorporated by reference herein in itsentirety.

In some embodiments, oligomers of the invention are functionalized atthe 5′ end in order to allow covalent attachment of the conjugatedmoiety to the 5′ end of the oligomer. In other embodiments, oligomers ofthe invention can be functionalized at the 3′ end. In still otherembodiments, oligomers of the invention can be functionalized along thebackbone or on the heterocyclic base moiety. In yet other embodiments,oligomers of the invention can be functionalized at more than oneposition independently selected from the 5′ end, the 3′ end, thebackbone and the base.

In some embodiments, activated oligomers of the invention aresynthesized by incorporating during the synthesis one or more monomersthat is covalently attached to a functional moiety. In otherembodiments, activated oligomers of the invention are synthesized withmonomers that have not been functionalized, and the oligomer isfunctionalized upon completion of synthesis.

In some embodiments, the oligomers are functionalized with a hinderedester containing an aminoalkyl linker, wherein the alkyl portion has theformula (CH₂)_(w), wherein w is an integer ranging from 1 to 10,preferably about 6, wherein the allyl portion of the alkylamino groupcan be straight chain or branched chain, and wherein the functionalgroup is attached to the oligomer via an ester group(—C(O)—(CH₂)_(w)NH).

In other embodiments, the oligomers are functionalized with a hinderedester containing a (CH₂)_(w)-sulfhydryl (SH) linker, wherein w is aninteger ranging from 1 to 10, preferably about 6, wherein the allportion of the alkylamino group can be straight chain or branched chain,and wherein the functional group attached to the oligomer via an estergroup (—O—C(O)—(CH₂)_(w)SH) In some embodiments, sulfhydryl-activatedoligonucleotides are conjugated with polymer moieties such aspolyethylene glycol or peptides (via formation of a disulfide bond).

Activated oligomers containing hindered esters as described above can besynthesized by any method known in the art, and in particular, bymethods disclosed in PCT Publication No. WO 2008/034122 and the examplestherein, which is incorporated herein by reference in its entirety.

Activated oligomers covalently linked to at least one functional moietycan be synthesized by any method known in the art, and in particular, bymethods disclosed in U.S. Patent Publication No. 2004/0235773, which isincorporated herein by reference in its entirety, and in Zhao et al.(2007) J. Controlled Release 119:143-152; and Zhao et al. (2005)Bioconjugate Chem. 16:758-766.

In still other embodiments, the oligomers of the invention arefunctionalized by introducing sulfhydryl, amino or hydroxyl groups intothe oligomer by means of a functionalizing reagent substantially asdescribed in U.S. Pat. Nos. 4,962,029 and 4,914,210, i.e., asubstantially linear reagent having a phosphoramidite at one end linkedthrough a hydrophilic spacer chain to the opposing end which comprises aprotected or unprotected sulfhydryl, amino or hydroxyl group. Suchreagents primarily react with hydroxyl groups of the oligomer. In someembodiments, such activated oligomers have a functionalizing reagentcoupled to a 5′-hydroxyl group of the oligomer. In other embodiments,the activated oligomers have a functionalizing reagent coupled to a3′-hydroxyl group. In still other embodiments, the activated oligomersof the invention have a functionalizing reagent coupled to a hydroxylgroup on the backbone of the oligomer. In yet further embodiments, theoligomer of the invention is functionalized with more tan one of thefunctionalizing reagents as described in U.S. Pat. Nos. 4,962,029 and4,914,210, incorporated herein by reference in their entirety. Methodsof synthesizing such functionalizing reagents and incorporating theminto monomers or oligomers are disclosed in U.S. Pat. Nos. 4,962,029 and4,914,210.

In some embodiments, the 5′-terminus of a solid-phase bound oligomer isfunctionalized with a dienyl phosphoramidite derivative, followed byconjugation of the deprotected oligomer with, e.g., an amino acid orpeptide via a Diels-Alder cycloaddition reaction.

In various embodiments, the incorporation of monomers containing2′-sugar modifications, such as a 2′-carbamate substituted sugar or a2′-(O-pentyl-N-phthalimido)-deoxyribose sugar into the oligomerfacilitates covalent attachment of conjugated moieties to the sugars ofthe oligomer. In other embodiments, an oligomer with an amino-containinglinker at the 2′-position of one or more monomers is prepared using areagent such as, for example, 5′dimethoxytrityl-2′-O-(e-phthalimidylaminopentyl)-2′-deoxyadenosine-3′—N,N-diisopropyl-cyanoethoxyphosphoramidite. See, e.g., Manoharan, et al., Tetrahedron Letters,1991, 34, 7171.

In still further embodiments, the oligomers of the invention haveamine-containing functional moieties on the nucleobase, including on theN6 purine amino groups, on the exocyclic N2 of guanine, or on the N4 or5 positions of cytosine. In various embodiments, such functionalizationmay be achieved by using a commercial reagent that is alreadyfunctionalized in the oligomer synthesis.

Some functional moieties are commercially available, for example,heterobifunctional and homobifunctional linking moieties are availablefrom the Pierce Co. (Rockford, Ill.). Other commercially availablelinking groups are 5′-Amino-Modifier C6 and 3′-Amino-Modifier reagents,both available from Glen Research Corporation (Sterling, Va.).5′-Amino-Modifier C6 is also available from ABI (Applied BiosystemsInc., Foster City, Calif.) as Aminolink-2, and 3′-Amino-Modifier is alsoavailable from Clontech Laboratories Inc. (Palo Alto, Calif.).

Compositions

In various embodiments, the oligomer of the invention is used inpharmaceutical formulations and compositions. Suitably, suchcompositions comprise a pharmaceutically acceptable diluent, carrier,salt or adjuvant. PCT/DK2006/000512 provides suitable and preferredpharmaceutically acceptable diluents, carriers and adjuvants—which arehereby incorporated by reference. Suitable dosages, formulations,administration routes, compositions, dosage forms, combinations withother therapeutic agents, pro-drug formulations are also provided inPCT/DK2006/000512—which are also hereby incorporated by reference.Details on techniques for formulation and administration also may befound in the latest edition of “REMINGTON'S PHARMACEUTICAL SCIENCES”(Maack Publishing Co, Easton Pa.).

In some embodiments, an oligomer of the invention is covalently linkedto a conjugated moiety to aid in delivery of the oligomer across cellmembranes. An example of a conjugated moiety that aids in delivery ofthe oligomer across cell membranes is a lipophilic moiety, such ascholesterol. In various embodiments, an oligomer of the invention isformulated with lipid formulations that form liposomes, such asLipofectamine 2000 or Lipofectamine RNAiMAX, both of which arecommercially available from Invitrogen. In some embodiments, theoligomers of the invention are formulated with a mixture of one or morelipid-like non-naturally occurring small molecules (“lipidoids”).Libraries of lipidoids can be synthesized by conventional syntheticchemistry methods and various amounts and combinations of lipidoids canbe assayed in order to develop a vehicle for effective delivery of anoligomer of a particular size to the targeted tissue by the chosen routeof administration. Suitable lipidoid libraries and compositions can befound, for example in Akinc et al. (2008) Nature Biotechnol., availableat http://www.nature.com/nbt/journal/vaop/ncurrent/abs/nbt1402.html,which is incorporated by reference herein.

As used herein, the term “pharmaceutically acceptable salts” refers tosalts that retain the desired biological activity of the hereinidentified compounds and exhibit acceptable levels of undesired toxiceffects. Non-limiting examples of such salts can be formed with organicamino acid and base addition salts formed with metal cations such aszinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt,nickel, cadmium, sodium, potassium, and the like, or with a cationformed from ammonia, N,N′-dibenzylethylene-diamine, D-glucosamine,tetraethylammonium, or ethylenediamine; or (c) combinations of (a) and(b); e.g., a zinc tannate salt or the like.

In certain embodiments, the pharmaceutical compositions according to theinvention comprise other active ingredients in addition to an oligomeror conjugate of the invention, including active agents useful for thetreatment of cancer, such as prostate cancer or breast cancer,particularly agents used in conventional antiandrogen therapy.

In some embodiments, additional active agents are non-steroidalantiandrogens (NSAAs), which block the binding of androgens at thereceptor site, or luteinizing hormone-releasing hormone analogues(LHRH-As) that suppress testicular production of androgens to castratelevels.

NSAAs such as CASODEX, when used with an LHRH-A as part of CombinedAndrogen Blockade therapy, help to inhibit the growth of prostate cancercells. In one embodiment, the invention provides for a combined androgenblockade therapy, characterised in that the therapy comprisesadministering the pharmaceutical composition according to the invention,and an NSAA and/or LHRH-A agent, which in certain embodiments areadministered prior to, during or subsequent to the administration of thepharmaceutical compositions of the invention.

The invention also provides a kit of parts wherein a first partcomprises at least one oligomer, conjugate and/or the pharmaceuticalcomposition according to the invention and a further part comprises anon-steroidal antiandrogen and/or a luteinizing hormone-releasinghormone analogue. It is therefore envisaged that the kit of parts may beused in a method of treatment, as referred to herein, where the methodcomprises administering both the first part and the further part, eithersimultaneously or one after the other.

Applications

The term “treatment” as used herein refers to both treatment of anexisting disease (e.g., a disease or disorder as referred to hereinbelow), or prevention of a disease, i.e., prophylaxis. It will thereforebe recognised that, in certain embodiments, “treatment” includesprophylaxis.

In various embodiments, the oligomers of the invention may be utilizedas research reagents for, for example, diagnostics, therapeutics andprophylaxis.

In some embodiments, such oligomers may be used for research purposes tospecifically inhibit the expression of androgen receptor protein(typically by degrading or inhibiting the AR mRNA and thereby preventingprotein formation) in cells and experimental animals, therebyfacilitating functional analysis of the target or an appraisal of itsusefulness as a target for therapeutic intervention.

In certain embodiments, the oligomers may be used in diagnostics todetect and quantitate androgen receptor expression in cells and tissuesby Northern blotting, in-situ hybridisation or similar techniques.

In various therapeutic embodiments, a non-human animal or a humansuspected of having a disease or disorder which can be treated bymodulating the expression of androgen receptor is treated byadministering an effective amount of an oligomer in accordance with thisinvention. Further provided are methods of treating a mammal, such astreating a human, suspected of having or being prone to a disease orcondition, associated with expression of androgen receptor byadministering a therapeutically or prophylactically effective amount ofone or more of the oligomers, conjugates or compositions of theinvention.

In certain embodiments, the invention also provides for the use of thecompounds or conjugates of the invention as described for themanufacture of a medicament for the treatment of a disorder as referredto herein, or for a method of the treatment of a disorder as referred toherein.

In various embodiments, the invention also provides for a method fortreating a disorder as referred to herein, said method comprisingadministering a compound according to the invention as herein described,and/or a conjugate according to the invention, and/or a pharmaceuticalcomposition according to the invention to a patient in need thereof.

Medical Indications

In certain therapeutic embodiments, the disorder to be treated iscancer, such as prostate cancer or breast cancer. In variousembodiments, the treatment of such a disease or condition according tothe invention may be combined with one or more other anti-cancertreatments, such as radiotherapy, chemotherapy or immunotherapy.

In certain other embodiments, the disorder to be treated is selectedfrom alopecia, benign prostatic hyperplasia, spinal and muscular atrophyand Kennedy disease and polyglutamate disease.

In various embodiments, the disease or disorder is associated with amutation of the AR gene or a gene whose protein product is associatedwith or interacts with AR. Therefore, in various embodiments, the targetmRNA is a mutated form of the AR sequence, for example, it comprises oneor more single point mutations or triplet repeats.

In other embodiments, the disease or disorder is associated withabnormal levels of a mutated form of androgen receptor. In variousembodiments, the disease or disorder is associated with abnormal levelsof a wild-type form of AR.

In various embodiments, the invention relates to methods of modulatingthe expression of the gene product of an androgen receptor target gene,i.e., a gene that is regulated by AR. Such AR receptor target geneproducts are selected form the group consisting of Protein kinase Cdelta (PRKCD), Glutathione S-transferase theta 2 (GSTT2), transientreceptor potential cation channel subfamily V member 3 (TRPV3),Pyrroline-5-carboxylate reductase 1 (PYCR1) and ornithineaminotransferase (OAT). In some embodiments, modulation of an AR targetgene results in increased expression or activity of the target gene. Inother embodiments, modulation of an AR target gene results in decreasedexpression or activity of the target gene.

The invention further provides use of a compound of the invention in themanufacture of a medicament for the treatment of any and all conditionsdisclosed herein.

In various embodiments, the invention is directed to a method oftreating a mammal suffering from or susceptible to a conditionassociated with abnormal levels of androgen receptor mRNA or protein,comprising administering to the mammal a therapeutically effectiveamount of an oligomer of the invention, or a conjugate thereof, thatcomprises one or more LNA monomers.

An interesting aspect of the invention is directed to the use of anoligomer (compound) as defined herein or a conjugate as defined hereinfor the preparation of a medicament for the treatment of a condition asdisclosed herein above.

In various embodiments, the invention encompasses a method of preventingor treating a disease comprising administering a therapeuticallyeffective amount of an oligomer according to the invention, or aconjugate thereof, to a human in need of such therapy.

In certain embodiments, the LNA oligomers of the invention, orconjugates thereof, are administered for a short period time rather thancontinuously.

In certain embodiments of the invention, the oligomer (compound) islinked to a conjugated moiety, for example, in order to increase thecellular uptake of the oligomer. In one embodiment the conjugated moietyis a sterol, such as cholesterol.

In various embodiments, the invention is directed to a method fortreating abnormal levels of androgen receptor, the method comprisingadministering an oligomer of the invention, or a conjugate or apharmaceutical composition thereof, to a patient in need of suchtreatment, and further comprising the administration of a furtherchemotherapeutic agent. In some embodiments, the chemotherapeutic agentis conjugated to the oligomer, is present in the pharmaceuticalcomposition, or is administered in a separate formulation.

The invention also relates to an oligomer, a composition or a conjugateas defined herein for use as a medicament.

The invention further relates to use of a compound, composition, or aconjugate as defined herein for the manufacture of a medicament for thetreatment of abnormal levels of androgen receptor or expression ofmutant forms of AR (such as allelic variants, such as those associatedwith one of the diseases referred to herein).

Moreover, in various embodiments, the invention relates to a method oftreating a subject suffering from a disease or condition selected fromcancer, such as breast cancer or prostate cancer, alopecia, benignprostatic hyperplasia, spinal and muscular atrophy, Kennedy disease andpolyglutamate disease, the method comprising the step of administering apharmaceutical composition as defined herein to the subject in needthereof.

Suitable dosages, formulations, administration routes, compositions,dosage forms, combinations with other therapeutic agents, pro-drugformulations are also provided in PCT/DK2006/000512—which is herebyincorporated by reference.

The invention also provides for a pharmaceutical composition comprisinga compound or a conjugate as herein described or a conjugate, and apharmaceutically acceptable diluent, carrier or adjuvant.PCT/DK2006/000512 provides suitable and preferred pharmaceuticallyacceptable diluents, carriers and adjuvants—which are herebyincorporated by reference.

EMBODIMENTS

The following embodiments of the invention may be used in combinationwith the other embodiments described herein.

1. An oligomer of between 10-50 nucleobases in length which comprises acontiguous nucleobase sequence of a total of between 10-50 nucleobases,wherein said contiguous nucleobase sequence is at least 80% homologousto a corresponding region of a nucleic acid which encodes a mammalianandrogen receptor.

2. The oligomer according to embodiment 1, wherein said oligomercomprises at least one LNA unit.

3. The oligomer according to embodiment 1 or 2, wherein the contiguousnucleobase sequence comprises no more than 3, such as no more than 2mismatches to the corresponding region of a nucleic acid which encodes amammalian androgen receptor.

4. The oligomer according to embodiment 3, wherein said contiguousnucleobase sequence comprises no more than a single mismatch to thecorresponding region of a nucleic acid which encodes a mammalianandrogen receptor.

5. The oligomer according to embodiment 4, wherein said contiguousnucleobase sequence comprises no mismatches, (i.e. is complementary to)the corresponding region of a nucleic acid which encodes a mammalianandrogen receptor.

6. The oligomer according to any one of embodiments 1-5, wherein thenucleobase sequence of the oligomer consists of the contiguousnucleobase sequence.

7. The oligomer according to any one of embodiments 1-6, wherein thenucleic acid which encodes a mammalian androgen receptor is the humanandrogen receptor nucleotide sequence such as SEQ ID No 1, or anaturally occurring allelic variant thereof.

8. The oligomer according to any one of embodiments 1-7, wherein thecontiguous nucleobase sequence is complementary to a correspondingregion of both the human androgen receptor nucleic acid sequence and anon-human mammalian androgen receptor nucleic acid sequence, such as themouse androgen receptor nucleic acid sequence.

9. The oligomer according to any one of embodiments 1 to 8, wherein thecontiguous nucleobase sequence comprises a contiguous subsequence of atleast 7, nucleobase residues which, when formed in a duplex with thecomplementary androgen receptor target RNA is capable of recruitingRNaseH.

10. The oligomer according to embodiment 9, wherein the contiguousnucleobase sequence comprises of a contiguous subsequence of at least 8,at least 9 or at least 10 nucleobase residues which, when formed in aduplex with the complementary androgen receptor target RNA is capable ofrecruiting RNaseH.

11. The oligomer according to any one of embodiments 9 or 10 whereinsaid contiguous subsequence is at least 9 or at least 10 nucleobases inlength, such as at least 12 nucleobases or at least 14 nucleobases inlength, such as 14, 15 or 16 nucleobases residues which, when formed ina duplex with the complementary androgen receptor target RNA is capableof recruiting RNaseH.

12. The oligomer according to embodiment any one of embodiments 1-11wherein said oligomer is conjugated with one or more non-nucleobasecompounds.

13. The oligomer according to any one of embodiments 1-12, wherein saidoligomer has a length of between 10-22 nucleobases.

14. The oligomer according to any one of embodiments 1-13, wherein saidoligomer has a length of between 12-18 nucleobases.

15. The oligomer according to any one of embodiments 1-14, wherein saidoligomer has a length of 14, 15 or 16 nucleobases.

16. The oligomer according to any one of embodiments 1-15, wherein saidcontinuous nucleobase sequence corresponds to a contiguous nucleotidesequence present in a nucleic acid sequence selected from the groupconsisting of SEQ ID NO 86-106.

17. The oligomer according to any one of embodiments 1-16, wherein theoligomer or contiguous nucleobase sequence comprises, or is selectedfrom a corresponding nucleobase sequence present in a nucleotidesequence selected from the group consisting of SEQ ID NO 2-22.

18. The oligomer according to any one of embodiments 1-17, wherein saidcontiguous nucleobase sequence comprises at least one affinity enhancingnucleotide analogue.

19. The oligomer according to embodiment 18, wherein said contiguousnucleobase sequence comprises a total of 2, 3, 4, 5, 6, 7, 8, 9 or 10affinity enhancing nucleotide analogues, such as between 5 and 8affinity enhancing nucleotide analogues.

20. The oligomer according to any one of embodiments 1-19 whichcomprises at least one affinity enhancing nucleotide analogue, whereinthe remaining nucleobases are selected from the group consisting of DNAnucleotides and RNA nucleotides, preferably DNA nucleotides.

21. The oligomer according to any one of embodiments 1-20, wherein theoligomer comprises of a sequence of nucleobases of formula, in 5′ to 3′direction, A-B-C, and optionally of formula A-B-C-D, wherein:

(a) consists or comprises of at least one nucleotide analogue, such as1, 2, 3, 4, 5 or 6 nucleotide analogues, preferably between 2-5nucleotide analogues, preferably 2, 3 or 4 nucleotide analogues, mostpreferably 2, 3 or 4 consecutive nucleotide analogues and;

(b) consists or comprises at least five consecutive nucleobases whichare capable of recruiting RNAseH (when formed in a duplex with acomplementary RNA molecule, such as the AR mRNA target), such as DNAnucleobases, such as 5, 6, 7, 8, 9, 10, 11 or 12 consecutive nucleobaseswhich are capable of recruiting RNAseH, or between 6-10, or between 7-9,such as 8 consecutive nucleobases which are capable of recruitingRNAseH, and;

(c) consists or comprises of at least one nucleotide analogue, such as1, 2, 3, 4, 5, or 6 nucleotide analogues, preferably between 2-5nucleotide analogues, such as 2, 3 or 4 nucleotide analogues, mostpreferably 2, 3 or 4 consecutive nucleotide analogues, and;

(d) when present, consists or comprises, preferably consists, of one ormore DNA nucleotide, such as between 1-3 or 1-2 DNA nucleotides.

22. The oligomer according to embodiment 21, wherein region A consistsor comprises of 2, 3 or 4 consecutive nucleotide analogues.

23. The oligomer according to any one of embodiments 21-22, whereinregion B consists or comprises of 7, 8, 9 or 10 consecutive DNAnucleotides or equivalent nucleobases which are capable of recruitingRNAseH when formed in a duplex with a complementary RNA, such as theandrogen receptor mRNA target.

24. The oligomer according to any one of embodiments 21-23, whereinregion C consists or comprises of 2, 3 or 4 consecutive nucleotideanalogues.

25. The oligomer according to any one of embodiments 21-24, whereinregion D consists, where present, of one or two DNA nucleotides.

26. The oligomer according to any one of embodiments 21-25, wherein:

(a) Consists or comprises of 3 contiguous nucleotide analogues;

(b) Consists or comprises of 7, 8, 9 or 10 contiguous DNA nucleotides orequivalent nucleobases which are capable of recruiting RNAseH whenformed in a duplex with a complementary RNA, such as the androgenreceptor mRNA target;

(c) Consists or comprises of 3 contiguous nucleotide analogues;

(d) Consists, where present, of one or two DNA nucleotides.

27. The oligomer according to embodiment 26, wherein the contiguousnucleobase sequence consists of 10, 11, 12, 13 or 14 nucleobases, andwherein;

-   -   (a) Consists of 1, 2 or 3 contiguous nucleotide analogues;    -   (b) Consists of 7, 8, or 9 consecutive DNA nucleotides or        equivalent nucleobases which are capable of recruiting RNAseH        when formed in a duplex with a complementary RNA, such as the        androgen receptor mRNA target;

(c) Consists of 1, 2 or 3 contiguous nucleotide analogues;

(d) Consists, where present, of one DNA nucleotide.

28. The oligomer according to anyone of embodiments 21-27, wherein Bcomprises at least one LNA nucleobase which is in the alpha-Lconfiguration, such as alpha-L-oxy LNA.

29. The oligomer according to any one of embodiments 1-28, wherein thenucleotide analogue(s) are independently or collectively selected fromthe group consisting of: Locked Nucleic Acid (LNA) units; 2′-O-alkyl-RNAunits, 2′-Me-RNA units, 2′-amino-DNA units, 2′-fluoro-DNA units, PNAunits, HNA units, and INA units.

30. The oligomer according to embodiment 29 wherein all the nucleotideanalogues(s) are LNA units.

31. The oligomer according to any one of embodiments 1-30, whichcomprises 1, 2, 3, 4, 5, 6, 7. 8. 9 or 10 LNA units such as between 2and 8 nucleotide LNA units.

32. The oligomer according to any one of the embodiments 29-31, whereinthe LNAs are independently selected from oxy-LNA, thio-LNA, andamino-LNA, in either of the beta-D and alpha-L configurations orcombinations thereof.

33. The oligomer according to embodiment 32, wherein the LNAs are allbeta-D-oxy-LNA.

34. The oligomer according to any one of embodiments 21-33, wherein thenucleotide analogues or nucleobases of regions A and C arebeta-D-oxy-LNA.

35. The oligomer according to any one of embodiments 1-34, wherein atleast one of the nucleobases present in the oligomer is a modifiednucleobase selected from the group consisting of 5-methylcytosine,isocytosine, pseudoisocytosine, 5-bromouracil, 5-propynyluracil,6-aminopurine, 2-aminopurine, inosine, diaminopurine, and2-chloro-6-aminopurine.

36. The oligomer according to any one of embodiments 1-35, wherein saidoligomer hybridises with a corresponding mammalian androgen receptormRNA with a T_(m) of at least 50° C.

37. The oligomer according to any one of embodiments 1-36, wherein saidoligomer hybridises with a corresponding mammalian androgen receptormRNA with a T_(m) of no greater than 80° C.

38. The oligomer according to any one of embodiments 1-37, wherein theinternucleoside linkages are independently selected from the groupconsisting of: phosphodiester, phosphorothioate and boranophosphate.

39. The oligomer according to embodiment 38, wherein the oligomercomprises at least one phosphorothioate internucleoside linkage.

40. The oligomer according to embodiment 39, wherein the internucleosidelinkages adjacent to or between DNA or RNA units, or within region B arephosphorothioate linkages.

41. The oligomer according to embodiment 39 or 40, wherein the linkagesbetween at least one pair of consecutive nucleotide analogues is aphosphodiester linkage.

42. The oligomer according to embodiment 39 or 407 wherein all thelinkages between consecutive nucleotide analogues are phosphodiesterlinkages.

43. The oligomer according to embodiment 42 wherein all theinternucleoside linkages are phosphorothioate linkages.

44. A conjugate comprising the oligomer according to any one of theembodiments 1-43 and at least one non-nucleotide or non-polynucleotidemoiety covalently attached to said compound.

45. A pharmaceutical composition comprising an oligomer as defined inany of embodiments 1-43 or a conjugate as defined in embodiment 44, anda pharmaceutically acceptable diluent, carrier, salt or adjuvant.

46. A pharmaceutical composition according to 45, wherein the oligomeris constituted as a pro-drug.

47. A pharmaceutical composition according to embodiment 45 or 46, whichfurther comprises a further therapeutic agent selected from the groupconsisting of: Non-steroidal Antiandrogens and Luteinizinghormone-releasing hormone analogues.

48. Use of an oligomer as defined in any one of the embodiments 1-43, ora conjugate as defined in embodiment 44, for the manufacture of amedicament for the treatment of a disease or disorder selected from thegroup consisting of: Cancer such as breast cancer or prostate cancer,alopecia, benign prostatic hyperplasia, spinal and muscular atrophy,Kennedy disease and polyglutamate disease.

49. An oligomer as defined in any one of the embodiments 1-43, or aconjugate as defined in embodiment 44, for use in the treatment of adisease or disorder selected from the group consisting of: Cancer suchas breast cancer or prostate cancer, alopecia, benign prostatichyperplasia, spinal and muscular atrophy, Kennedy disease andpolyglutamate disease.

50. A method for treating a disease or disorder selected from the groupconsisting of: Cancer such as breast cancer or prostate cancer,alopecia, benign prostatic hyperplasia, spinal and muscular atrophy,Kennedy disease and polyglutamate disease, said method comprisingadministering an oligomer as defined in one of the embodiments 1-43, ora conjugate as defined in embodiment 44, or a pharmaceutical compositionas defined in any one of the embodiments 45-47, to a patient in needthereof.

51. A method for treating an cancer such as prostate cancer or breastcancer, said method comprising administering an oligomer as defined inone of the embodiments 1-43, or a conjugate as defined in embodiment 44,or a pharmaceutical composition as defined in any one of the embodiments45-47, to a patient in need thereof.

52. A method of reducing or inhibiting the expression of androgenreceptor in a cell or a tissue, the method comprising the step ofcontacting said cell or tissue with a compound as defined in one of theembodiments 1-43, or a conjugate as defined in embodiment 44, or apharmaceutical composition as defined in any one of the embodiments45-47, so that expression of androgen receptor is reduce or inhibited.

A method for modulating the expression of a gene which is regulated bythe androgen receptor (i.e. an androgen receptor target) in a cell whichis expressing said gene, said method comprising the step of contactingsaid cell or tissue with a compound as defined in one of the embodiments1-43, or a conjugate as defined in embodiment 44, or a pharmaceuticalcomposition as defined in any one of the embodiments 45-47, so thatexpression of androgen receptor target is modulated.

EXAMPLES Example 1 Monomer Synthesis

The LNA monomer building blocks and derivatives were prepared followingpublished procedures and references cited therein—see WO07/031,081 andthe references cited therein.

Example 2 Oligonucleotide Synthesis

Oligonucleotides were synthesized according to the method described inWO07/031,081. Table 1 shows examples of sequences of antisenseoligonucleotides of the invention. Tables 2 and 3 show examples ofantisense oligonucleotides (oligomers) of the invention.

Example 3 Design of the Oligonucleotides

In accordance with the invention, a series of oligomers were designed totarget different regions of human androgen receptor mRNA (GenBankAccession number NM_(—)000044; SEQ ID NO: 1).

SEQ ID NOS: 2-22, shown in Table 1, below, are sequences of oligomersdesigned to target human androgen receptor mRNA. The target region ofthe target nucleic acid is indicated in the table.

TABLE I Antisense Oligonucleotide Sequences Length Target site SEQ ID NOSequence (5′-3′) (bases) NM_000044 SEQ ID NO: 2 GAGAACCATCCTCACC 161389-1404 SEQ ID NO: 3 GGACCAGGTAGCCTGT 16 1428-1443 SEQ ID NO: 4CCCCTGGACTCAGATG 16 1881-1896 SEQ ID NO: 5 GCACAAGGAGTGGGAC 16 1954-1969SEQ ID NO: 6 GCTGTGAAGAGAGTGT 16 2422-2437 SEQ ID NO: 7 TTTGACACAAGTGGGA16 2663-2678 SEQ ID NO: 8 GTGACACCCAGAAGCT 16 2813-2828 SEQ ID NO: 9CATCCCTGCTTCATAA 16 2975-2990 SEQ ID NO: 10 ACCAAGTTTCTTCAGC 163008-3023 SEQ ID NO: 11 CTTGGCCCACTTGACC 16 3263-3278 SEQ ID NO: 12TCCTGGAGTTGACATT 16 3384-3399 SEQ ID NO: 13 CACTGGCTGTACATCC 163454-3469 SEQ ID NO: 14 CATCCAAACTCTTGAG 16 3490-3505 SEQ ID NO: 15GCTTTCATGCACAGGA 16 3529-3544 SEQ ID NO: 16 GAAGTTCATCAAAGAA 163594-3609 SEQ ID NO: 17 AGTTCCTTGATGTAGT 16 3616-3631 SEQ ID NO: 18TTGCACAGAGATGATC 16 3809-3824 SEQ ID NO: 19 GATGGGCTTGACTTTC 163845-3860 SEQ ID NO: 20 CAGGCAGAAGACATCT 16 3924-3939 SEQ ID NO: 21CCCAAGGCACTGCAGA 16 3960-3975 SEQ ID NO: 22 GCTGACATTCATAGCC 163114-3129 SEQ ID NO: 86 TGGGGAGAACCATCCTCAGCCTGC 24 1385-1408 SEQ ID NO:87 TCCAGGACCAGGTAGCCTGTGGGG 24 1424-1447 SEQ ID NO: 88TGTTCCCCTGGACTCAGATGCTCC 24 1877-1990 SEQ ID NO: 89TGGGGCACAAGGAGTGGGAGGCAC 24 1950-1973 SEQ ID NO: 90TTCGGCTGTGAAGAGAGTGTGCCA 24 2418-2441 SEQ ID NO: 91CGCTTTTGACACAAGTGGGACTGG 24 2659-2682 SEQ ID NO: 92CATAGTGACACCCAGAAGCTTCAT 24 2809-2832 SEQ ID NO: 93GAGTCATCCCTGCTTCATAACATT 24 2971-2994 SEQ ID NO: 94GATTACCAAGTTTCTTCAGCTTCC 24 3004-3027 SEQ ID NO: 95AGGCCTTGGCCCACTTGACCACGT 24 3259-3282 SEQ ID NO: 96AGCATCCTGGAGTTGACATTGGTG 24 3380-3403 SEQ ID NO: 97GACACACTGGCTGTACATCCGGGA 24 3450-3473 SEQ ID NO: 98GAGCCATCCAAACTCTTGAGAGAG 24 3486-3509 SEQ ID NO: 99CAGTGCTTTCATGCACAGGAATTC 24 3525-3548 SEQ ID NO: 100ATTCGAAGTTCATCAAAGAATTTT 24 3590-3613 SEQ ID NO: 101ATCGAGTTCCTTGATGTAGTTCAT 24 3612-3635 SEQ ID NO: 102GCACTTGCACAGAGATGATCTCTG 24 3805-3828 SEQ ID NO: 103AATAGATGGGCTTGACTTTCCCAG 24 3841-3864 SEQ ID NO: 104ATAACAGGCAGAAGACATCTGAAA 24 3920-3943 SEQ ID NO: 105ATTCCCCAAGGCACTGCAGAGGAG 24 3956-3979 SEQ ID NO: 106ATGGGCTGACATTCATAGCCTTCA 24 3110-3133

In SEQ ID NOs: 23-43, shown below in Table 2, upper case, boldfaceletters indicate nucleotide analogue monomers (e.g., •-D-oxy LNAmonomers) and subscript “s” represents phosphorothiote linkage groupsbetween the monomers. The absence of a subscript “s” (if any) indicatesa phosphodiester linkage group. Lower case letters represent DNAmonomers.

TABLE 2 Oligonucleotide designs SEQ ID NO Sequence (5′-3′) SEQ ID NO: 235′-G _(s) A _(s) G_(s)a_(s)a_(s)c_(s)c_(s)a_(s)t_(s)c_(s)c_(s)t_(s)c_(s) A _(s) C _(s)C-3′ SEQ ID NO: 24 5′-G _(s) G _(s) A_(s)c_(s)c_(s)a_(s)g_(s)g_(s)t_(s)a_(s)g_(s)c_(s)c_(s) T _(s) G _(s)T-3′ SEQ ID NO: 25 5′-C _(s) C _(s) C_(s)c_(s)t_(s)g_(s)g_(s)a_(s)c_(s)t_(s)c_(s)a_(s)g_(s) A _(s) T _(s)G-3′ SEQ ID NO: 26 5′-G _(s) C _(s) A_(s)c_(s)a_(s)a_(s)g_(s)g_(s)a_(s)g_(s)t_(s)g_(s)g_(s) G _(s) A _(s)C-3′ SEQ ID NO: 27 5′-G _(s) C _(s) T_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) T _(s) G _(s)T-3′ SEQ ID NO: 28 5′-T _(s) T _(s) T_(s)g_(s)a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)g_(s) G _(s) G _(s)A-3′ SEQ ID NO: 29 5′-G _(s) T _(s) G_(s)a_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)g_(s)a_(s)a_(s) G _(s) C _(s)T-3′ SEQ ID NO: 30 5′-C _(s) A _(s) T_(s)c_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)c_(s)a_(s) T _(s) A _(s)A-3′ SEQ ID NO: 31 5′-A _(s) C _(s) C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s) A _(s) G _(s)C-3′ SEQ ID NO: 32 5′-C _(s) T _(s) T_(s)g_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)t_(s)t_(s)g_(s) A _(s) C _(s)C-3′ SEQ ID NO: 33 5′-T _(s) C _(s) C_(s)t_(s)g_(s)g_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)c_(s) A _(s) T _(s)T-3′ SEQ ID NO: 34 5′-C _(s) A _(s) C_(s)t_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)c_(s)a_(s) T _(s) C _(s)C-3′ SEQ ID NO: 35 5′-C _(s) A _(s) T_(s)c_(s)c_(s)a_(s)a_(s)a_(s)c_(s)t_(s)c_(s)t_(s)t_(s) G _(s) A _(s)G-3′ SEQ ID NO: 36 5′-G _(s) C _(s) T_(s)t_(s)t_(s)c_(s)a_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s) G _(s) G _(s)A-3′ SEQ ID NO: 37 5′-G _(s) A _(s) A_(s)g_(s)t_(s)t_(s)c_(s)a_(s)t_(s)c_(s)a_(s)a_(s)a_(s) G _(s) A _(s)A-3′ SEQ ID NO: 38 5′-A _(s) G _(s) T_(s)t_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s) A _(s) G _(s)T-3′ SEQ ID NO: 39 5′-T _(s) T _(s) G_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s) A _(s) T _(s)C-3′ SEQ ID NO: 40 5′-G _(s) A _(s) T_(s)g_(s)g_(s)g_(s)c_(s)t_(s)t_(s)g_(s)a_(s)c_(s)t_(s) T _(s) T _(s)C-3′ SEQ ID NO: 41 5′-C _(s) A _(s) G_(s)g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s) T _(s) C _(s)T-3′ SEQ ID NO: 42 5′-C _(s) C _(s) C_(s)a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)t_(s)g_(s)c_(s) A _(s) G _(s)A-3′ SEQ ID NO: 43 5′-G _(s) C _(s) T_(s)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)a_(s) G _(s) C _(s)C-3′

Example 4 In Vitro Model: Cell Culture

The effect of antisense oligonucleotides on target nucleic acidexpression can be tested in any of a variety of cell types provided thatthe target nucleic acid is present at measurable levels. The target canbe expressed endogenously or by transient or stable transfection of anucleic acid encoding said target. The expression level of targetnucleic acid can be routinely determined using, for example, Northernblot analysis, Real-Time PCR, Ribonuclease protection assays. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen.

Cells were cultured in the appropriate medium as described below andmaintained at 37° C. at 95-98% humidity and 5% CO₂. Cells were routinelypassaged 2-3 times weekly.

A549 The human lung cancer cell line A5439 was cultured in DMEM(Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25μg/ml).

MCF7 The human breast cancer cell line MCF7 was cultured in EagleMEM(Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+1×NEAA+gentamicin(25 μg/ml).

Example 5 In Vitro Model: Treatment with Antisense Oligonucleotide

The cell lines listed in Example 4 were treated with an oligomer usingthe cationic liposome formulation LipofectAMINE 2000 (Gibco) astransfection vehicle. Cells were seeded in 6-well cell culture plates(NUNC) and treated when 80-90% confluent. Oligomer concentrations usedranged from 1 nM to 16 nM final concentration. Formulation ofoligomer-lipid complexes were carried out essentially as described bythe manufacturer using serum-free OptiMEM (Gibco) and a final lipidconcentration of 5 μg/mL LipofectAMINE 2000. Cells were incubated at 37°C. for 4 hours and treatment was stopped by removal ofoligomer-containing culture medium. Cells were washed andserum-containing media was added. After oligomer treatment, cells wereallowed to recover for 20 hours before they were harvested for RNAanalysis.

Example 6 In Vitro Model: Extraction of RNA and cDNA Synthesis

Total RNA Isolation and First Strand Synthesis

Total RNA was extracted from cells transfected as described above andusing the Qiagen RNeasy kit (Qiagen cat. no. 74104) according to themanufacturer's instructions. First strand synthesis was performed usingReverse Transcriptase reagents from Ambion according to themanufacturer's instructions.

For each sample, the volume of 0.5 •g total RNA was adjusted to 10.8 •lwith RNase free H₂O and mixed with 2 •l random decamers (50 •M) and 4 •ldNTP mix (2.5 mM each dNTP) and heated to 70° C. for 3 min, after whichthe samples were rapidly cooled on ice. After cooling the samples onice, 2 •l 10× Buffer RT, 1 •l MMLV Reverse Transcriptase (100 U/•l) and0.25 •l RNase inhibitor (10 U/•l) were added to each sample, followed byincubation at 42° C. for 60 min, heat inactivation of the enzyme at 95°C. for 10 min and then cooling of the sample to 4° C.

Example 7 In Vitro Model: Analysis of Oligonucleotide Inhibition ofAndrogen Receptor Expression by Real-Time PCR

Antisense modulation of androgen receptor expression can be assayed in avariety of ways known in the art. For example, androgen receptor mRNAlevels can be quantitated by, e.g., Northern blot analysis, competitivepolymerase chain reaction (PCR), or real-time PCR. Real-timequantitative PCR is presently preferred. RNA analysis can be performedon total cellular RNA or mRNA.

Methods of RNA isolation and RNA analysis such as Northern blot analysisare routine in the art and are taught in, for example, Current Protocolsin Molecular Biology, John Wiley and Sons.

Real-time quantitative (PCR) can be conveniently accomplished using thecommercially available Multi-Color Real Time PCR Detection System,available from Applied Biosystems.

Real-Time Quantitative PCR Analysis of Androgen Receptor mRNA Levels

The amount of human androgen receptor mRNA in the samples was quantifiedusing the human androgen receptor ABI Prism Pre-Developed TaqMan AssayReagents (Applied Biosystems cat. no. Hs00171172_m1) according to themanufacturer's instructions.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA quantity was usedas an endogenous control for normalizing any variance in samplepreparation.

The amount of human GAPDH mRNA in the samples was quantified using thehuman GAPDH ABI Prism Pre-Developed TaqMan Assay Reagent (AppliedBiosystems cat. no. 4310884E) according to the manufacturer'sinstructions.

Real-time Quantitative PCR is a technique well known in the art and istaught in for example Heid et al. Real time quantitative PCR, GenomeResearch (1996), 6: 986-994.

Real Time PCR

The cDNA from the first strand synthesis performed as described inExample 6 was diluted 2-20 times, and analyzed by real time quantitativePCR using Taquman 7500 FAST or 7900 FAST from Applied Biosystems. Theprimers and probe were mixed with 2× Taqman Fast Universal PCR mastermix (2×) (Applied Biosystems Cat. #4364103) and added to 4 μl cDNA to afinal volume of 10 μl. Each sample was analysed in duplicate. Standardcurves were generated by assaying 2-fold dilutions of a cDNA that hadbeen prepared on material purified from a cell line expressing the RNAof interest. Sterile H₂O was used instead of cDNA for the no-templatecontrol. PCR program: 95° C. for 30 seconds, followed by 40 cycles of95° C., 3 seconds, 60° C., 20-30 seconds. Relative quantities of targetmRNA were determined from the calculated Threshold cycle using theApplied Biosystems Fast System SDS Software Version 1.3.1.21. or SDSSoftware Version 2.3.

Example 8 In Vitro Analysis: Antisense Inhibition of Human AndrogenReceptor mRNA Expression by Oligonucleotide Compounds

Oligonucleotides presented in Table 3 were evaluated for their potentialto knock down androgen receptor mRNA expression at concentrations of 1,4 and 16 nM (see FIGS. 1 and 2).

The data in Table 3 are presented as percentage down-regulation relativeto mock transfected cells at 16 nM. Lower case letters represent DNAmonomers, bold, upper case letters represent β-D-oxy-LNA monomers. Allcytosine bases in the LNA monomers are 5-methylcytosines. Subscript “s”represents a phosphorothioate linkage.

TABLE 3 Inhibition of human androgen receptor mRNA expression byoligonucleotides Percent Percent inhibition inhibition of of AndrogenAndrogen Test substance Sequence (5′-3′) receptor - MCF7 receptor - A549SEQ ID NO: 44 5′-G _(s) A _(s) G_(s)a_(s)a_(s)c_(s)c_(s)a_(s)t_(s)c_(s)c_(s)t_(s)c_(s) A _(s) C _(s)C-3′ 80.1 63.8 SEQ ID NO: 45 5′-G _(s) G _(s) A_(s)c_(s)c_(s)a_(s)g_(s)g_(s)t_(s)a_(s)g_(s)c_(s)c_(s) T _(s) G _(s)T-3′ 89.0 88.2 SEQ ID NO: 46 5′-C _(s) C _(s) C_(s)c_(s)t_(s)g_(s)g_(s)a_(s)c_(s)t_(s)c_(s)a_(s)g_(s) A _(s) T _(s)G-3′ 89.4 82.8 SEQ ID NO: 47 5′-G _(s) C _(s) A_(s)c_(s)a_(s)a_(s)g_(s)g_(s)a_(s)g_(s)t_(s)g_(s)g_(s) G _(s) A _(s)C-3′ 83.1 77.7 SEQ ID NO: 48 5′-G _(s) C _(s) T_(s)g_(s)t_(s)g_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) T _(s) G _(s)T-3′ 93.8 96.7 SEQ ID NO: 49 5′-C _(s) T _(s) G_(s)t_(s)g_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s)g_(s) T _(s) G-3′ n.d. n.d.SEQ ID NO: 50 5′-T _(s) G _(s)t_(s)g_(s)a_(s)a_(s)g_(s)a_(s)g_(s)a_(s) G_(s) T _(s)-3′ n.d. n.d. SEQ ID NO: 51 5′-T _(s) T _(s) T_(s)g_(s)a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)g_(s) G _(s) G _(s)A-3′ 96.9 95.5 SEQ ID NO: 52 5′-T _(s) T _(s) G_(s)a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s)g_(s) G _(s) G-3′ n.d. n.d.SEQ ID NO: 53 5′-T _(s) G _(s)a_(s)c_(s)a_(s)c_(s)a_(s)a_(s)g_(s)t_(s) G_(s) G-3′ n.d. n.d. SEQ ID NO: 54 5′-G _(s) T _(s) G_(s)a_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)g_(s)a_(s)a_(s) G _(s) C _(s)T-3′ 95.4 98.3 SEQ ID NO: 55 5′-T _(s) G _(s) A_(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)g_(s)a_(s)a_(s) G _(s) C-3′ n.d. n.d.SEQ ID NO: 56 5′-G _(s) A _(s)c_(s)a_(s)c_(s)c_(s)c_(s)a_(s)g_(s)a_(s) A_(s) G-3′ n.d. n.d. SEQ ID NO: 57 5′-C _(s) A _(s) T_(s)c_(s)c_(s)c_(s)t_(s)g_(s)c_(s)t_(s)t_(s)c_(s)a_(s) T _(s) A _(s)A-3′ 89.5 88.9 SEQ ID NO: 58 5′-A _(s) C _(s) C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s) A _(s) G _(s)C-3′ 95.6 98.9 SEQ ID NO: 59 5′-C _(s) C _(s) A_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s) A _(s) G-3′ n.d. n.d.SEQ ID NO: 60 5′-C _(s) A _(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s) C_(s) A-3′ n.d. n.d. SEQ ID NO: 61 5′-C _(s) T _(s) T_(s)g_(s)g_(s)c_(s)c_(s)c_(s)a_(s)c_(s)t_(s)t_(s)g_(s) A _(s) C _(s)C-3′ 86.7 93.3 SEQ ID NO: 62 5′-T _(s) C _(s) C_(s)t_(s)g_(s)g_(s)a_(s)g_(s)t_(s)t_(s)g_(s)a_(s)c_(s) A _(s) T _(s)T-3′ 81.3 93.0 SEQ ID NO: 63 5′-C _(s) A _(s) C_(s)t_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)c_(s)a_(s) T _(s) C _(s)C-3′ 90.9 98.4 SEQ ID NO: 64 5′-A _(s) C _(s) T_(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)c_(s)a_(s) T _(s) C-3′ n.d. n.d.SEQ ID NO: 65 5′-C _(s) T _(s)g_(s)g_(s)c_(s)t_(s)g_(s)t_(s)a_(s)c_(s) A_(s) T-3′ n.d. n.d. SEQ ID NO: 66 5′-C _(s) A _(s) T_(s)c_(s)c_(s)a_(s)a_(s)a_(s)c_(s)t_(s)c_(s)t_(s)t_(s) G _(s) A _(s)G-3′ 79.8 95.3 SEQ ID NO: 67 5′-G _(s) C _(s) T_(s)t_(s)t_(s)c_(s)a_(s)t_(s)g_(s)c_(s)a_(s)c_(s)a_(s)g_(s) G _(s) A-3′83.5 97.0 SEQ ID NO: 68 5′-G _(s) A _(s) A_(s)g_(s)t_(s)t_(s)c_(s)a_(s)t_(s)c_(s)a_(s)a_(s)a_(s) G _(s) A _(s)A-3′ 88.2 85.6 SEQ ID NO: 69 5′-A _(s) G _(s) T_(s)t_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)t_(s)g_(s)t_(s) A _(s) G _(s)T-3′ 92.7 94.0 SEQ ID NO: 70 5′-G _(s) T _(s) T_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)t_(s)g_(s) T _(s) A _(s) G-3′ n.d.n.d. SEQ ID NO: 71 5′-T _(s) T_(s)c_(s)c_(s)t_(s)t_(s)g_(s)a_(s)t_(s)g_(s) T _(s) A-3′ n.d. n.d. SEQID NO: 72 5′-T _(s) T _(s) G_(s)c_(s)a_(s)c_(s)a_(s)g_(s)a_(s)g_(s)a_(s)t_(s)g_(s) A _(s) T _(s)C-3′ 79.2 90.4 SEQ ID NO: 73 5′-G _(s) A _(s) T_(s)g_(s)g_(s)g_(s)c_(s)t_(s)t_(s)g_(s)a_(s)c_(s)t_(s) T _(s) T _(s)C-3′ 91.1 97.3 SEQ ID NO: 74 5′-A _(s) T_(s)g_(s)g_(s)g_(s)c_(s)t_(s)t_(s)g_(s)a_(s)c_(s)t_(s) T _(s) T-3′ n.d.n.d. SEQ ID NO: 75 5′-T _(s) G_(s)g_(s)g_(s)c_(s)t_(s)t_(s)g_(s)a_(s)c_(s) T _(s) T-3′ n.d. n.d. SEQID NO: 76 5′-C _(s) A _(s) G_(s)g_(s)c_(s)a_(s)g_(s)a_(s)a_(s)g_(s)a_(s)c_(s)a_(s) T _(s) C _(s)T-3′ 85.9 94.3 SEQ ID NO: 77 5′-C _(s) C _(s) C_(s)a_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)t_(s)g_(s)c_(s) A _(s) G _(s)A-3′ 93.0 98.5 SEQ ID NO: 78 5′-C _(s) C _(s) A_(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)t_(s)g_(s)c_(s) A _(s) G-3′ n.d. n.d.SEQ ID NO: 79 5′-C _(s) A _(s)a_(s)g_(s)g_(s)c_(s)a_(s)c_(s)t_(s)g_(s) C_(s) A-3′ n.d. n.d. SEQ ID NO: 80 5′-G _(s) C _(s) T_(s)g_(s)a_(s)c_(s)a_(s)t_(s)t_(s)c_(s)a_(s)t_(s)a_(s) G _(s) C _(s)C-3′ n.d. n.d.

As shown in Table 3, oligonucleotides having the sequences set forth inSEQ ID NOs: 48, 51, 54, 58, 63, 69, 73 and 77 at 16 nM demonstratedgreater than 90% inhibition of androgen receptor mRNA expression in A549and MCF7 cells in these experiments.

In certain embodiments, oligomers based on the tested antisense oligomersequences and designs, but having, for example, different lengths(shorter or longer) and/or monomer content (e.g. the type and/or numberof nucleoside analogues) than those shown, e.g., in Table 3, could alsoprovide suitable inhibition of androgen receptor expression.

Example 9 In Vivo Analysis: Antisense Inhibition of Mouse AndrogenReceptor mRNA Liver Expression by Oligonucleotide Compounds

Nude mice were dosed i.v. q3dx4 with 100 mg/kg oligonucleotide (groupsize of 5 mice). The antisense oligonucleotides (SEQ ID:48, SEQ ID:51,SEQ ID:58, SEQ ID:63, SEQ ID:77) were dissolved in phosphate bufferedsaline. Animals were sacrificed 24 h after last dosing and liver tissuewas sampled and stored in RNA later until RNA extraction and QPCRanalysis. Total RNA was extracted and AR mRNA expression in liversamples was measured by QPCR as described in Example 7 using a mouse ARQPCR assay (cat. Mm01238475_m1, Applied Biosystems). Results werenormalised to mouse GAPDH (cat. no. 4352339E, Applied Biosystems) andknock-own was quantitated relative to saline treated controls. The datain Table 4 are presented as percentage down-regulation relative tosaline treated animals.

TABLE 4 In vivo knock-down of AR mRNA expression Compound Liver (% KD)Saline 0 SEQ ID: 51 100 mg/kg  65.0 +/− 12.6 SEQ ID: 58 100 mg/kg 95.2+/− 1.0 SEQ ID: 77 100 mg/kg 91.9 +/− 3.9

As shown in Table 4, oligonucleotides of SEQ ID NOs: 58 and 77 at 100mg/kg demonstrated greater than 90% inhibition of androgen receptor mRNAexpression in mouse liver cells in these experiments.

Example 10 In vitro analysis: Antisense Exhibition of Human Androgenreceptor mRNA

Measurement of Proliferating Viable Cells (MTS Assay)

LNCaP prostate cancer and A549 lung cancer cells were seeded to adensity of 150,000 cells per well in a 6-well plate the day prior totransfection. A549 cells were cultured in DMEM (Sigma)+10% fetal bovineserum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml) whereas LNCaP cellswere cultured in RPMI 1640 Medium (Sigma)+10% fetal bovine serum (FBS)+2mM Glutamax I+gentamicin (25 μg/ml). On the following day, medium wasremoved followed by addition of 1.2 ml OptiMEM containing 5 μg/mlLipofectamine2000 (Invitrogen). Cells were incubated for 7 min beforeadding 0.3 ml oligonucleotides diluted in OptiMEM. The finaloligonucleotide concentrations were 4 nM and 16 nM. After 4 hours oftreatment, media was removed and cells were trypsinized and seeded to adensity of 5000 cells per well in a clear 96 well plate (ScientificOrange no. 1472030100) in 100 μl media. Viable cells were measured atthe times indicated by adding 10 μl the tetrazolium compound[3-(4,5-dimethyl-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt; MTS] and an electron coupling reagent (phenazineethosulfate; PES) (CellTiter 96® AQueous One Solution Cell ProliferationAssay, Promega). Viable cells were measured at 490 nm in a Powerwave(Biotek Instruments). The OD490 nm measurements were plotted againsttime/h. (See FIG. 13 and FIG. 14). As shown in FIG. 13 and FIG. 14,oligonucleotides of SEQ ID NOs: 58 and 77 inhibit growth of both LNCaPprostate and A549 lung cancer cells.

Example 11 In Vitro Analysis: Caspase 3/7 Activity by AntisenseInhibition of Human Androgen Receptor mRNA

LNCaP prostate cancer cells and A549 lung cancer cells were seeded to adensity of 150,000 cells per well in a 6-well plate the day prior totransfection. A549 cells were cultured in DMEM (Sigma)+10% fetal bovineserum (FBS)+2 mM Glutamax I+gentamicin (25 μg/ml) whereas LNCaP cellswere cultured in RPMI 1640 Medium (Sigma)+10% fetal bovine serum CUBS)+2mM Glutamax I+gentamicin (25 μg/ml). The next day medium was removedfollowed by addition of 1.2 ml OptiMEM containing 5 μg/mlLipofectamine2000 (Invitrogen). Cells were incubated for 7 min beforeadding 0.3 ml oligonucleotides diluted in OptiMEM. The finaloligonucleotide concentrations were 4 nM and 16 nM. After 4 hours oftreatment, media was removed and cells were trypsinized and seeded to adensity of 5000 cells per well in a white 96 well plate (Nunc) in 100 μlmedia. Caspase 3/7 activity was measured at the times indicated byadding 100 μl Caspase-Glo 3/7 assay (Promega). Caspase 3/7 activity wasmeasured using a luminometer. The caspase 3/7 activities were measuredat three different time points 14 h, 48 h and 72 h (See FIG. 15 and FIG.16). As shown in FIG. 15 and FIG. 16, oligonucleotides of SEQ ID NOs: 58and 77 induce caspase 3/7 activity in both LNCaP prostate and A549 lungcancer cells.

Example 12 In Vitro Analysis: Antisense Inhibition of Human AndrogenReceptor mRNA Expression by Oligonucleotide Compounds in Prostate CancerCell Line LNCaP and Lung Cancer Cell Line A549

Oligonucleotides were evaluated for their potential to knock downandrogen receptor mRNA expression at concentrations of 0.5, 1, 2, 4, 8and 16 nM (see FIG. 11). LNCaP prostate cancer cells and A549 lungcancer cells were seeded to a density of 150,000 cells per well in a6-well plate the day prior to transfection. A549 cells were cultured inDMEM (Sigma)+10% fetal bovine serum (FBS)+2 mM Glutamax I+gentamicin (25μg/ml). LNCaP cells were cultured in RPMI 1640 Medium (Sigma)+10% fetalbovine serum (FBS)+2 mM Glutamax I+gentamicin (25 μg/nu). On thefollowing day, medium was removed followed by addition of 1.2 ml OptiMEMcontaining 5 μg/ml Lipofectamine2000 (Invitrogen). Cells were incubatedfor 7 min before adding 0.3 ml oligonucleotides diluted in OptiMEM. Thefinal oligonucleotide concentrations were 0.5, 1, 2, 4, 8 and 16 nM.Cells were washed and serum-containing media was added. After oligomertreatment cells were allowed to recover for 20 hours before they wereharvested for RNA analysis. The procedure for RNA isolation, cDNAsynthesis and qPCR were as described in Examples 5, 6 and 7. As shown inFIGS. 11 and 12 oligonucleotides of SEQ ID NOs: 58 and 77 were potent inknocking down AR mRNA expression in both the lung cancer cell line A549and in the androgen receptor-dependent LNCaP prostate cancer cell line.

Example 13 In Vivo Analysis: Effect of Antisense Oligonucleotides on PSALevels and Androgen-Dependent Prostate Tumor Growth in Mice

Six to seven week old male athymic nu/nu mice (Harlan Sprague Dawley)weighing an average of 27.3±2.4 g were used in the study. Ten millioncells of 22RV1 (androgen-independent prostate cancer line) weresuspended in PBS (Gibco#14190) and Matrigel (BD#356234) with a ratio of1:1 were injected subcutaneously into each mouse. When tumors reached anaverage volume of 150-200 mm³, the mice were divided into nineexperimental groups. Two hundred μl of oligomer were injectedintravenously when the average tumor size reached 152.66±27.97 mm³.Oligomers were given every 3 days for a total of 5 dosings. The controlvehicles were given using the same dosing regimen as the oligomers. Onday 16, mice were sacrificed and blood collected in EDTA laced tubes andspun for 5 min. 50 μl of the supernatants were then subjected to PSAassay using the ELISA kit from ALPCO Diagnostics in Salem (PSAHU-L01).Results of the experiment are shown in FIG. 17.

Six to seven week old male athymic nu/nu mice (Harlan Sprague Dawley)weighing an average of 27.3±2.4 g were used in the study. Ten millioncells of 22RV1 (androgen-independent prostate cancer line) weresuspended in PBS (Gibco#14190) and Matrigel (BD#356234) with a ratio of1:1 were injected subcutaneously into each mouse. When tumors reached anaverage volume of 150-200 mm³, the mice were divided into nineexperimental groups. Two hundred μl of oligomer was injectedintravenously when the average tumor size reached 152.66±27.97 mm³Oligomers were given every 3 days for a total of 5 dosings. The controlvehicles were given using the same dosing regimen as the oligomers. Thetumor volumes for each mouse were determined by measuring two dimensionswith calipers and calculated using the formula: tumorvolume=(length×width²)/2). Results of the experiment are shown in FIG.18.

Example 14 Preparation of Conjugates of Oligomers with PolyethyleneGlycol

The oligomers having sequences shown as SEQ ID NO: 48 or SEQ ID NO: 63are functionalized on the 5′ terminus by attaching an aminoalkyl group,such as hexan-1-amine blocked with a blocking group such as Fmoc to the5′ phosphate groups of the oligomers using routine phosphoramiditechemistry, oxidizing the resultant compounds, deprotecting them andpurifying them to achieve the functionalized oligomers, respectively,having the formulas (IA) and (IB):

wherein the bold uppercase letters represent nucleoside analoguemonomers, lowercase letters represent DNA monomers, the subscript “s”represents a phosphorothioate linkage, and ^(Me)C represents5-methylcytosine.

A solution of activated PEG, such as the one shown in formula (II):

wherein the PEG moiety has an average molecular weight of 12,000, andeach of the compounds of formulas (IA) and (IB) in PBS buffer arestirred in separate vessels at room temperature for 12 hours. Thereaction solutions are extracted three times with methylene chloride andthe combined organic layers are dried over magnesium sulphate andfiltered and the solvent is evaporated under reduced pressure. Theresulting residues are dissolved in double distilled water and loadedonto an anion exchange column. Unreacted PEG linker is eluted with waterand the products are eluted with NH₄HCO₃ solution. Fractions containingpure products are pooled and lypophilized to yield the conjugates SEQ IDNOs: 48 and 63, respectively as show in formulas (IIIA) and (IIIB):

wherein each of the oligomers of SEQ ID NOs: 48 and 63 is attached to aPEG polymer having average molecular weight of 12,000 via a releasablelinker.

Chemical structures of PEG polymer conjugates that can be made witholigomers having sequences shown in SEQ ID NOs: 51, 58 and 77 using theprocess described above are respectively shown in formulas (IVA), (IVB)and (IVC):

wherein bold uppercase letters represent beta-D-oxy-LNA monomers,lowercase letters represent DNA monomers, the subscript “s” represents aphosphorothioate linkage and ^(Me)C represent 5-methylcytosine.

Activated oligomers that can be used in this process to respectivelymake the conjugates shown in formulas (IVA), (IVB) and (IVC) have thechemical structures shown in formulas (VA), (VB) and (VC):

1. An oligomer consisting of the formula: (SEQ ID NO: 58) 5′-A_(s)^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base.
 2. A conjugate comprising theoligomer of claim 1 covalently attached to at least one moiety that isnot a nucleic acid or a monomer.
 3. A pharmaceutical compositioncomprising: the oligomer of claim 1 or a conjugate comprising saidoligomer covalently attached to at least one moiety that is not anucleic acid or a monomer; and a pharmaceutically acceptable diluent,carrier, salt or adjuvant.
 4. A method of inhibiting the expression ofandrogen receptor in a cell, comprising: contacting said cell with aneffective amount of an oligomer consisting of the formula (SEQ ID NO:58) 5′-A_(s) ^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base.
 5. A method of inhibiting theexpression of androgen receptor in a cell, comprising contacting saidcell with an effective amount of a conjugate according to claim
 2. 6. Amethod of inhibiting the expression of androgen receptor in a tissue ofa mammal, comprising: contacting said tissue with an effective amount ofan oligomer consisting of the formula (SEQ ID NO: 58) 5′-A_(s)^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base.
 7. A method of inhibiting theexpression of androgen receptor in a tissue of a mammal comprisingcontacting said tissue with an effective amount of a conjugate accordingto claim
 2. 8. A method of inhibiting the expression of an androgenreceptor target gene in a cell or tissue of a mammal, comprising:contacting said cell or tissue with an effective amount of an oligomerconsisting of the formula (SEQ ID NO: 58) 5′-A_(s) ^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base.
 9. A method of treating breastcancer or prostate cancer in a mammal, comprising; administering to saidmammal an effective amount of an oligomer consisting of the formula (SEQID NO: 58) 5′-A_(s) ^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base.
 10. A method of treating a disorderin a mammal, comprising: administering to said mammal an effectiveamount of an oligomer consisting of the formula (SEQ ID NO: 58) 5′-A_(s)^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base, wherein said disorder is selectedfrom the group consisting of alopecia, benign prostatic hyperplasia,spinal and muscular atrophy, Kennedy disease, and polyglutamate disease.11. An activated oligomer comprising: an oligomer consisting of theformula (SEQ ID NO: 58) 5′-A_(s) ^(Me)C_(s)^(Me)C_(s)a_(s)a_(s)g_(s)t_(s)t_(s)t_(s)c_(s)t_(s)t_(s)c_(s)A_(s)G_(s)^(Me)C-3′,

wherein uppercase letters denote beta-D-oxy-LNA monomers and lowercaseletters denote DNA monomers, the subscript “s” denotes aphosphorothioate linkage, and ^(Me)C denotes a beta-D-oxy-LNA monomercontaining a 5-methylcytosine base; and at least one functional groupcovalently attached thereto at one or more positions independentlyselected from the 5′-end, the 3′ end, the 2′-OH of a ribose sugar, andthe base.